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Plant Immunity Methods and Protocols 1st Edition
Patrick Cournoyer Digital Instant Download
Author(s): Patrick Cournoyer, S. P. Dinesh-Kumar (auth.), John M. McDowell
(eds.)
ISBN(s): 9781617379970, 1617379972
Edition: 1
File Details: PDF, 5.78 MB
Year: 2011
Language: english

Me t h o d s i n Mo l e c u l a r Bi o l o g y ™
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Hatfield, Hertfordshire, AL10 9AB, UK
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Plant Immunity
Methods and Protocols
Edited by
John M. McDowell
Department of Plant Pathology, Physiology, and Weed Science,
Virginia Tech, Blacksburg, VA, USA

Editor
John M. McDowell, Ph.D.
Department of Plant Pathology,
Physiology, and Weed Science
Virginia Tech
Latham Hall (0390) 550
Blacksburg, VA 24061, USA
johnmcd@vt.edu
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61737-997-0 e-ISBN 978-1-61737-998-7
DOI 10.1007/978-1-61737-998-7
Library of Congress Control Number: 2011921723
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
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v
Preface
Examples of How New Experimental Technologies Have Enabled Landmark
Advances in Understanding of Plant Immunity Over the Last Half-Century
This volume of Methods in Molecular Biology was designed to emphasize emerging
technologies that can be applied to outstanding questions in plant immunity. The content
is complementary to another recent, excellent volume in the series with a similar focus (1).
Below, I provide a brief historical overview highlighting major conceptual advances in
molecular plant–microbe interactions that would not have been possible without exploita-
tion of new technologies. Additionally, I outline current conceptual challenges in our field
that can be addressed with methods described in this volume. Finally, I speculate on tech-
nological advances in the near term that enable deeper understanding of plant immunity
and support rational strategies for durable disease control.
As all readers of this volume know, much effort has been invested in understanding
the molecular mechanisms through which plants and microbes interact. Much of the
progress in this field has been fueled by timely, thoughtful exploitation of new methodolo-
gies. For example, H.H. Flor’s use of classical genetics clearly demonstrated that the out-
come of encounters between flax and flax rust can be dictated by single genes on both
sides of the interaction (2). Equally important, his methodology revealed striking specific-
ity in these interactions, which led to formulation of the seminal “gene-for-gene” model.
This genetics-driven model provided a conceptual framework for the plant immunity that
proved generally applicable and remains relevant today (3, 4).
Subsequent emergence of molecular biology tools enabled the gene-for-gene model
to be elaborated in molecular terms. For example, gene cloning technologies were used to
isolate avirulence (avr) genes, resistance (R) genes, and additional components of patho-
genicity and immunity. Molecular approaches, along with judicious biochemistry, pro-
vided for critical examination of “receptor-ligand” models that predicted direct interaction
between the products of R and avr genes (e.g., (5)). Three important themes emerged
from these efforts: First, the majority of plant resistance genes encode proteins from a
single superfamily, defined by a nucleotide-binding site and leucine-rich repeats (NB-LRR)
(6). Second, pathogen Avr proteins are, in many cases, translocated into plant cells where
they act as effectors to reprogram plant cells for susceptibility (7). Third, NB-LRR pro-
teins often do not interact directly with corresponding Avr proteins but instead monitor
guardees or decoys that are modified by the Avr protein (8–10). In addition, experiments
with pathogen “elicitor” molecules revealed a second branch of the plant immune system,
which directly recognizes pathogen-associated molecular patterns (PAMPs) that are evo-
lutionarily conserved among diverse pathogens (11). The two branches of the plant
immune system have been connected by recent models predicting that pathogen effectors
may have evolved to interfere with PAMP-triggered immunity (12). NB-LRR receptors
thereby provide a second line of defense by recognizing the molecular signatures of

vi Preface
effector activity. In sum, the adoption of molecular biology methods led to major advances
in understanding of plant immunity that could not have been anticipated by models based
(however logically) on genetic data alone.
At present, “omics” tools are being used to build on molecular advances and provide
new insights. For example, it is now possible to survey a plant genome and identify all of
the potential immune receptors using queries based on conserved motifs (e.g., (13)).
From this, it has become clear that plants maintain hundreds of probable immune recep-
tors, which in many cases appear to be evolving dynamically to cope with ever-changing
pathogen populations (14). From an applied perspective, these inventories can greatly
accelerate the process of resistance gene identification (e.g., for cloning and/or marker-
assisted breeding of R genes from wild relatives into crops).
Similar advances are underway in pathogen genomics. For example, molecular signa-
tures are being developed for pathogen effector proteins that enable comprehensive effec-
tor gene inventories to be predicted in silico. Genome level comparisons have revealed that
pathogen genomes contain dozens (in prokaryotes) to hundreds (in eukaryotes) of effec-
tor genes (15, 16). Like the cognate surveillance genes in plants, these genes are often
variable and subject to rapid turnover. Large-scale characterization of effector functions is
a major focus of effort in the field of plant immunity that is discussed further below.
Transcript profiling is also impacting understanding of plant immunity. For example,
early experiments with microarrays revealed massive transcriptional changes that accom-
pany the activation of the immune system and illuminated molecular distinctions between
different resistance mechanisms (e.g., (17)). Subsequent studies that combine transcript
profiling with immune response mutants have provided insight into the structure of the
defense hierarchy and have identified previously unknown components of the network
(e.g., (18)). Analyses of transcript profiles have also provided important insights into the
molecular mechanisms through which pathogens manipulate the environment inside plant
tissue (e.g., (19)).
At present, it is inarguable that our current, exciting level of understanding of plant
immunity (and pathogen evasion thereof) owes much to the timely adoption of new meth-
odologies in genetics and molecular biology, as well as genomics. However, we remain far
from a complete understanding of how the plant immune system functions, or how its
functionality is perturbed by adapted pathogens. Many questions remain that will require
new methodologies to be developed, optimized, and widely adopted. This volume of
Methods in Molecular Biology was designed to emphasize emerging technologies that can
be applied to outstanding questions in plant immunity.
For example, although NB-LRR immune surveillance proteins have now been known
of for 1.5 decades, we still do not understand exactly how they function, and it is not
clear whether all NB-LRR proteins function in a similar manner (20, 21). Moreover, we
still lack a complete inventory of downstream signaling components, and we do not
understand how these components interact. Methods that can be applied for new insights
into molecular functionality of NB-LRR proteins and other immune signaling compo-
nents are described in Chaps.1–4. Chapter 1 addresses the understudied question of
exactly where in the cell NB-LRR proteins exercise their functions of surveillance and
downstream activation. In particular, the approaches therein can be applied to study
dynamic relocalization of NB-LRRs in response to pathogen invasion (e.g., (22)).
Chapter 2 describes a very innovative “fragment complementation” approach for under-
standing the functions of intramolecular interactions between different NB-LRR func-
tional domains (e.g., (23)). Chapters 3 and 4 provide new protocols for the oft-vexing

vii
Preface
process of purifying low-abundance protein complexes. These protocols were developed with
the immediate goal of identifying the components within immune surveillance complexes
(e.g., (24, 25)), but are also potentially applicable to any protein expressed in planta.
Chapters 5–7 have similarly broad applicability. Chapter 5 describes chromatin immu-
noprecipitation, which is being widely used to characterize protein–DNA interactions
in vivo and identify targets of transcription factors in a variety of organisms (e.g., (26)).
This chapter was written in reference to WRKY transcription factors, which are ubiquitous
in plants and are key regulators of immunity and other plant processes. The procedures
could be adapted for other plant proteins (e.g., NB-LRR proteins that function inside the
nucleus) or for pathogen effector proteins that mimic plant transcription factors. Chapter 6
provides new information on an inducible system for plant transgene expression that is
frequently used in studies of plant immunity (e.g., for expressing effector proteins in
planta (27)). This chapter helps researchers maximize the versatility of this system and
clearly understand its limitations. Chapter 7 describes a creative method for detection and
quantification of alternatively spliced transcripts (28). Alternative splicing is important for
the regulation of some NB-LRR resistance gene regulation and is currently understudied
with respect to immune system function (29).
Chapters 8–17 describe methods used to identify and functionally characterize patho-
gen effector proteins. As mentioned above, pathogen genomics have revealed a plethora
of candidate effectors. Understanding how they function is one of the most active areas in
plant–pathogen research at present (15, 16). One emergent generality is that almost all
types of pathogens deploy moderate to large batallions of secreted effectors, many of
which operate inside plant cells. Chapters 9 and 10 provide approaches to isolate plant
cells that are in intimate contact with fungi and nematodes, respectively. These cells can
serve as sources for cDNA libraries that are enriched for transcripts encoding effectors
(e.g., (30, 31)). This is a proven approach toward effector gene discovery for pathogens
with no reference genome sequence.
The bacterium Pseudomonas syringae has been at the forefront of effector characteriza-
tion, and Chap.10 describes methods whereby single or multiple gene knockouts can be
constructed. This approach is vital to establish loss-of-function phenotypes, deconvolute the
redundancy in effector repertoires, and evaluate the contribution of effectors to bacterial
host range (e.g., (32)). In the eukaryotic kingdom, oomycetes from the Phytophthora genus
have been at the forefront of effector identification; however, transformation of Phytophthora
is often challenging even for experienced labs (33). Chapter 11 provides procedures for
transformation of P. capcisi, which appears more amenable to genetic manipulation and can
infect N. benthamiana and defense-compromised Arabidopsis mutants. Chapter 12 describes
procedures pertaining to a second oomycete, Hyaloperonsopora arabidopsidis, that has long
been used as a model pathogen of Arabidopsis and is becoming even more widely used for
oomycete comparative genomes and investigation of oomycete effector proteins (34).
Bacteria deploy dozens of effectors, and oomycetes, fungi, and nematodes likely
produce many-fold more (15, 16). To facilitate functional characterization of large col-
lections of effectors, several high-throughput assays have been recently developed. Two
such assays, presented in Chaps. 13 and 14, can be used to estimate immune-suppres-
sive capacity of effectors from almost any pathogen (e.g., (35–37)). Chapter 15
describes a transient expression system optimized for protein complex purification,
similar to Chaps. 3 and 4, that can be applied at medium-throughput to identify plant
proteins which interact with pathogen effectors (or other types of protein interactions
in planta). Chapters 16 and 17 describe approaches for visualizing subcellular localization

viii Preface
of effectors in plant cells, which is of key importance for understanding effector function
(e.g., (38)).
In the final chapters, the focus returns to the plant at a fine spatial scale. One aspect of
plant–pathogen interactions that has not been adequately addressed relates to spatial dif-
ferences in the molecular responses of plant cells in different locations of the infected
organ, relative to pathogen infection structures. Chapters 18–20 provide information on
laser microdissection, which is one of the most promising technologies for addressing
questions relating to spatio-temporal differences in different cell types in infected organs
(e.g., (39)). Finally, Chap.21 zooms in even further (completing a spatial circle with
Chap.1) to describe exciting approaches to visualize subcellular dynamics in infected cells
(e.g., (40)). This is undoubtedly one of the major emerging areas in plant–microbe inter-
actions in the upcoming years (41).
The authors of these chapters sincerely hope that our contributions are of use, and we
wish readers the best of success in applying these methods to their favorite pathosystems. We
also look forward to the next volume(s) in this series that address plant–microbe interac-
tions. Perhaps the next volume describes new technologies for structural studies of immune
receptor complexes, along with advanced proteomic and metabolomic surveys of infected
tissue at fine spatial scales. A major challenge will be to integrate data from disparate
approaches, with the potential payoff being holistic models of infected cells, tissues, and
organs. It would be particularly valuable to understand regulatory connections between
immunity and other plant processes that might predict undesirable side effects of engineered
resistance strategies (e.g., yield loss, reduced resistance to abiotic stress). It is exciting to
imagine that such depth of understanding might even prompt a subsequent Methods vol-
ume focusing on “translational” approaches; for example, bioinformatic approaches to effi-
ciently identify durable resistance genes for breeding or transgenics, or even surveillance
genes that are custom-designed to detect PAMPs or indispensable pathogen effectors. Is this
far-fetched? Perhaps…but if we were plant breeders in the 1950s could we have anticipated
the depth of understanding that has already been achieved in only five short decades?
Blackburg, VA John M. McDowell
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xi
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Studying NB-LRR Immune Receptor Localization by Agroinfiltration
Transient Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Patrick Cournoyer and S.P. Dinesh-Kumar
2 Fragment Complementation and Co-immunoprecipitation Assays
for Understanding R Protein Structure and Function . . . . . . . . . . . . . . . . . . . . . . 9
Peter Moffett
3 Purification of Resistance Protein Complexes Using a Biotinylated
Affinity (HPB) Tag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Yiping Qi and Fumiaki Katagiri
4 Biochemical Purification of Native Immune Protein Complexes . . . . . . . . . . . . . . 31
James M. Elmore and Gitta Coaker
5 Chromatin Immunoprecipitation to Identify Global Targets of WRKY
Transcription Factor Family Members Involved in Plant Immunity . . . . . . . . . . . . 45
Mario Roccaro and Imre E. Somssich
6 Dose–Response to and Systemic Movement of Dexamethasone
in the GVG-Inducible Transgene System in Arabidopsis . . . . . . . . . . . . . . . . . . . . 59
Xueqing Geng and David Mackey
7 Quantifying Alternatively Spliced mRNA via Capillary Electrophoresis . . . . . . . . . 69
Xue-Cheng Zhang and Walter Gassmann
8 Constructing Haustorium-Specific cDNA Libraries from Rust Fungi . . . . . . . . . . 79
Ann-Maree Catanzariti, Rohit Mago, Jeff Ellis, and Peter Dodds
9 Microaspiration of Esophageal Gland Cells and cDNA Library Construction
for Identifying Parasitism Genes of Plant-Parasitic Nematodes . . . . . . . . . . . . . . . 89
Richard S. Hussey, Guozhong Huang, and Rex Allen
10 Construction of Pseudomonas syringae pv. tomato DC3000 Mutant
and Polymutant Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Brian H. Kvitko and Alan Collmer
11 A Straightforward Protocol for Electro-transformation of Phytophthora
capsici Zoospores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Edgar Huitema, Matthew Smoker, and Sophien Kamoun
12 Propagation, Storage, and Assays with Hyaloperonospora arabidopsidis:
A Model Oomycete Pathogen of Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
John M. McDowell, Troy Hoff, Ryan G. Anderson, and Daniel Deegan
13 Assaying Effector Function in Planta Using Double-Barreled
Particle Bombardment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Shiv D. Kale and Brett M. Tyler

xii Contents
14 Assays for Effector-Mediated Suppression of Programmed Cell Death in Yeast . . . 173
Yuanchao Wang and Qian Huang
15 Purification of Effector–Target Protein Complexes via Transient Expression
in Nicotiana benthamiana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Joe Win, Sophien Kamoun, and Alexandra M. E. Jones
16 Imaging Fluorescently Tagged Phytophthora Effector Proteins
Inside Infected Plant Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Petra C. Boevink, Paul R.J. Birch, and Stephen C. Whisson
17 Immunolocalization of Pathogen Effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Eric Kemen, Kurt Mendgen, and Ralf T. Voegele
18 Laser Capture Microdissection of Nematode Feeding Cells . . . . . . . . . . . . . . . . . 227
Nagabhushana Ithal and Melissa G. Mitchum
19 Laser Microdissection of Plant–Fungus Interaction Sites and Isolation
of RNA for Downstream Expression Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Divya Chandran, Noriko Inada, and Mary C. Wildermuth
20 Global Expression Profiling of RNA from Laser Microdissected Cells
at Fungal–Plant Interaction Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Divya Chandran, Greg Hather, and Mary C. Wildermuth
21 Visualizing Cellular Dynamics in Plant–Microbe Interactions
Using Fluorescent-Tagged Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
William Underwood, Serry Koh, and Shauna C. Somerville
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

xiii
Contributors
Rex Allen • Department of Plant Pathology, University of Georgia, Athens, GA, USA
Ryan G. Anderson • Department of Plant Pathology, Physiology, and Weed Science,
Paul R. J. Birch • Division of Plant Sciences, College of Life Sciences, University
of Dundee at SCRI, Invergowrie, Dundee, UK
Petra C. Boevink • Plant Pathology Programme, Scottish Crop Research Institute,
Invergowrie, Dundee, UK
Ann-Maree Catanzariti • Department of Plant and Microbial Biology, University
of California, Berkeley, CA, USA
Divya Chandran • Department of Plant and Microbial Biology, University
Gitta Coaker • Department of Plant Pathology, University of California, Davis, CA,
USA
Alan Collmer • Department of Plant Pathology and Plant-Microbe Biology,
Cornell University, Ithaca, NY, USA
Patrick Cournoyer • Department of Molecular, Cellular, and Developmental
Biology, Yale University, New Haven, CT, USA
Daniel Deegan • Department of Plant Pathology, Physiology, and Weed Science,
S. P. Dinesh-Kumar • Department of Molecular, Cellular, and Developmental
Biology, Yale University, New Haven, CT, USA
Peter Dodds • Division of Plant Industry, Commonwealth Scientific and Industrial
Research Organisation, Canberra, ACT, Australia
Jeff Ellis • Division of Plant Industry, Commonwealth Scientific and Industrial
James M. Elmore • Department of Plant Pathology, University of California,
Davis, CA, USA
Walter Gassmann • Division of Plant Sciences, Christopher S. Bond Life Sciences
Center, University of Missouri, Columbia, MO, USA
Xueqing Geng • Department of Horticulture and Crop Science,
The Ohio State University, Columbus, OH, USA;
Department of Plant Cellular and Molecular Biology, The Ohio State University,
Columbus, OH, USA
Greg Hather • Department of Plant and Microbial Biology,
University of California, Berkeley, CA, USA;
Department of Statistics, University of California, Berkeley, CA, USA
Troy Hoff • Department of Plant Pathology, Physiology, and Weed Science,

xiv Contributors
Guozhong Huang • Department of Plant Pathology, University of Georgia,
Athens, GA, USA
Qian Huang • Plant Pathology Department, Nanjing Agricultural University,
Nanjing, China
Edgar Huitema • The Sainsbury Laboratory, John Innes Centre, Norwich, UK
Richard S. Hussey • Department of Plant Pathology, University of Georgia,
Athens, GA, USA
Noriko Inada • Department of Plant and Microbial Biology, University
Nagabhushana Ithal • Division of Plant Sciences, Bond Life Sciences Center,
University of Missouri, Columbia, MO, USA
Alexandra M. E. Jones • The Sainsbury Laboratory, John Innes Centre,
Norwich, UK
Shiv D. Kale • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
Sophien Kamoun • The Sainsbury Laboratory, John Innes Centre, Norwich, UK
Fumiaki Katagiri • Department of Plant Biology, University of Minnesota,
St. Paul, MN, USA
Eric Kemen • The Sainsbury Laboratory, John Innes Centre, Norwich, UK
Serry Koh • Plant Systems Engineering Research Center, Korea Research Institute
of Bioscience and Biotechnology, Daejeon, Republic of Korea
Brian H. Kvitko • Department of Plant Pathology and Plant-Microbe Biology,
Cornell University, Ithaca, NY, USA
David Mackey • Department of Horticulture and Crop Science,
The Ohio State University, Columbus, OH, USA
Department of Plant Cellular and Molecular Biology, The Ohio State University,
Columbus, OH, USA
Rohit Mago • Division of Plant Industry, Commonwealth Scientific and Industrial
John M. McDowell • Department of Plant Pathology, Physiology, and Weed Science,
Kurt Mendgen • Phytopathologie, Fachbereich Biologie, Universität Konstanz,
Konstanz, Germany
Melissa G. Mitchum • Division of Plant Sciences, Bond Life Sciences Center,
University of Missouri, Columbia, MO, USA
Peter Moffett • Boyce Thompson Institute for Plant Research, Ithaca, NY, USA
Yiping Qi • Department of Plant Biology, University of Minnesota, St. Paul, MN, USA
Mario Roccaro • Department of Plant Microbe Interaction, Max Planck Institute
for Plant Breeding, Cologne, Germany
Matthew Smoker • The Sainsbury Laboratory, John Innes Centre, Norwich, UK
Shauna C. Somerville • Department of Plant and Microbial Biology, Energy
Biosciences Institute, University of California, Berkeley, CA, USA
Imre E. Somssich • Department of Plant Microbe Interaction, Max Planck Institute
for Plant Breeding, Cologne, Germany
Brett M. Tyler • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg,
VA, USA

xv
Contributors
William Underwood • Energy Biosciences Institute, University of California,
Berkeley, CA, USA
Ralf T. Voegele • Phytopathologie, Fachbereich Biologie, Universität Konstanz,
Konstanz, Germany
Yuanchao Wang • Plant Pathology Department, Nanjing Agricultural University,
Nanjing, China
Stephen C. Whisson • Plant Pathology Programme, Scottish Crop Research Institute,
Invergowrie, Dundee, UK
Mary C. Wildermuth • Department of Plant and Microbial Biology, University
Joe Win • The Sainsbury Laboratory, John Innes Centre, Norwich, UK
Xue-Cheng Zhang • Division of Plant Sciences, Christopher S. Bond Life Sciences
Center, University of Missouri, Columbia, MO, USA

1
John M. McDowell (ed.), Plant Immunity: Methods and Protocols, Methods in Molecular Biology, vol. 712,
DOI 10.1007/978-1-61737-998-7_1, © Springer Science+Business Media, LLC 2011
Chapter 1
Studying NB-LRR Immune Receptor Localization
by Agroinfiltration Transient Expression
Patrick Cournoyer and S.P. Dinesh-Kumar
Abstract
NB-LRR immune receptors in plants play dual roles as sentries and as activators of defense. The site in
the cell where these activities take place can be different for different NB-LRRs. Furthermore, recogni-
tion and defense activation can occur in distinct subcellular compartments. Therefore, determining the
subcellular localization of NB-LRRs is a key step toward understanding how they function. Recent
advances in confocal microscopy enable high-resolution imaging of proteins in live cells. Agroinfiltration
in the Nicotiana benthamiana model plant system is a convenient way of expressing proteins for localiza-
tion studies. This chapter explains how to use N. benthamiana to transiently express NB-LRRs for confo-
cal fluorescence microscopy.
Key words: Localization, NB-LRR, Confocal microscopy, R gene, Agroinfiltration, Nicotiana,
Transient expression
The plant innate immune system uses two tiers of defenses to

protect plants from pathogens. In the first, cell-surface receptors
recognize conserved molecular patterns shared by many microbes
and induce basal immune responses. When pathogens circumvent
or block basal immunity, intracellular NB-LRR immune receptors
act as a second line of defense. NB-LRRs recognize specific patho-
gen effectors present in the cell. To be effective, NB-LRRs must
reside where pathogen effectors localize or where they exert their
influence. They must also reside at sites where they can promote
signals that lead to defense activation. NB-LRR localization is
therefore an area of research that is critical to understand how these
receptors function. Biochemical fractionation has been used for
investigating subcellular localization of NB-LRRs. Although
1. Introduction

2 Cournoyer and Dinesh-Kumar

biochemicalfractionationisinformative,fluorescentprotein
tagging
combined with confocal fluorescence microscopy provides the
advantage of seeing the protein’s location and movement in live
cells, in real time, and in three dimensions.
Microscopy has been used to study the localization of several
NB-LRRs to-date. The N immune receptor from Nicotiana was
shown to be nuclear and cytoplasmic, even though it does not
possess a canonical nuclear localization signal (NLS). N’s pres-
ence in the nucleus was shown to be required for function (1).
The barley immune receptor MLA10 also lacks a canonical NLS
and was shown to be nuclear and cytoplasmic. Like N, MLA10
nuclear localization is required for its function (2). Biochemical
fractionation experiments showed that MLA1, a related NB-LRR,
is present in the nucleus and appears to accumulate there at higher
levels during a defense response (2).
RRS1-R from Arabidopsis is an unusual NB-LRR because it
has a WRKY transcription factor domain and a canonical NLS. In
the presence of its cognate pathogen effector, it was observed in
the nucleus (3). Arabidopsis RPS4 is another NB-LRR that has a
canonical NLS. Microscopy analyses have shown that RPS4 local-
izes to the nucleus and to areas of the cytoplasm (4). Its NLS is
required for its nuclear localization and for its function.
Biochemical fractionation showed that RPS4 associates with
endomembranes and co-fractionates with an endoplasmic reticu-
lum marker (4). RPP1-A and RPM1 are other NB-LRRs from
Arabidopsis that are known to be membrane associated from

biochemical fractionation experiments (5, 6). Microscopy could
be used to examine the localization of these membrane-associated
NB-LRRs more closely.
Based on the NB-LRRs examined to date, it is clear that

different NB-LRRs can have different localization patterns. It is
also notable that NB-LRRs sometimes have localizations that
could not be predicted based on their sequence. Furthermore, it
is plausible that NB-LRRs move between different compart-
ments in the cell either constitutively or upon activation. Careful
examination using confocal fluorescence microscopy has great
potential to inform us about how NB-LRRs detect pathogens
and trigger defense.
Expressing a protein of interest fused to a fluorescent protein
in live cells is now routinely used in the study of protein localiza-
tion. Many factors, however, must be considered when deciding
on a strategy. The goal of any localization study is to replicate as
closely as possible the protein’s natural state. The best strategy
depends on the transformation capabilities for the plant system
under investigation. Agrobacterium-mediated transfection meth-
ods are favored for most dicot plants while particle bombardment
is commonly used to transfect monocot cells.

3
Studying NB-LRR Immune Receptor Localization
Agroinfiltration permits the rapid transformation of leaf cells
without the need to recover transgenic lines. Agroinfiltrated leaf
cells are ready for imaging 30–48 h after infiltration, compared to
months required for generating transgenic lines. Imaging is

possible before the onset of RNA silencing, which can be an
obstacle for protein expression in transgenic lines. It is also

possible to co-express multiple proteins in the same cells without
difficulty. These advantages have made agroinfiltration a popular
choice for localization studies.
Agroinfiltration is most commonly used to transfect leaves of
Nicotiana tabacum and Nicotiana benthamiana. Some plants are
not amenable to agroinfiltration because of unfavorable leaf

architecture or because of defense mechanisms that protect them
from Agrobacterium in leaves. Nicotiana is often used to study
the localization of immune receptors from other taxa because of
its convenience. While still informative, there are caveats to heter-
ologous expression. NB-LRRs rarely function outside of their
native plant family and therefore may or may not exhibit native
localization patterns. Heterologous expression may also lead to
spurious activation of programed cell death (PCD) as in the case
of RPS4 expression in Nicotiana (7). In Arabidopsis leaves,
Agrobacterium activates the EF-Tu receptor (EFR), resulting in
basal defense responses that prevent efficient transfection (8).
Arabidopsis efr mutants are amenable to agroinfiltration. Thus,
localization of Arabidopsis NB-LRRs can be studied by transient
expression without the need for a heterologous system.
Generating transgenic lines expressing fluorescent protein fusions
is the best way to test protein localization when agroinfiltration is
not possible. Transgenic lines are also useful for confirming obser-
vations in agroinfiltrated leaves. Monocots are generally recalci-
trant to Agrobacterium-mediated transformation and are often
transformed by particle bombardment. For localization studies,
transient expression by particle bombardment is possible (see ref. 2
for an example). Below, we focus on expression using agroinfiltra-
tion in Nicotiana benthamiana.
1. Cloned NB-LRR of interest.
2. Fluorescent protein fusion expression vector.
1. Nicotiana benthamiana seeds.
2. 0.1% agarose, autoclaved.
3. Growing materials: Potting mix, pots, growth facilities.
1.1. Agroinfiltration
1.2. Other Methods
of Expressing Proteins
for Localization
2. Materials
2.1. Creating an
Expression Vector
2.2. Growing Nicotiana

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the air, swinging left and right, full of such sounds as a cane chair makes.
Michael, squat and broad, patch on his eye, was telling some experience of
his life to somebody; another man drew near to listen and remained; still
more clustered round. Twosomes, talking in corners, desisted to listen also.
Michael! one of them called. Wasn't there something about you and
stowing away?
Oh, that's an old story, answered Michael.
What's that about? asked several of the younger men, who wanted to
gather as much data as possible on this subject. Tell us about it, Michael,
they besought him.
Well, said Michael, it was when I came over on the A-Chiles.
Was Johnson boss then?
Oh, before Johnson's time. I've been over with Johnson, too, said
Michael.
Shut up! several admonished. Let him tell the story.
I was on the rocks, said Michael. You see I got down to the docks too
late to get the A-Chiles back.
There was a movement of interest, a drawing closer. This was a
predicament they understood. There are always cattle to bring eastward
from Canadian and U.S. ports to Liverpool or London, and the cattleman
may return with his boss on the same ship; but if he loses it there are not
cattle-boats plying west across the Atlantic to give him a job again. There is
stoking to be done, of course, east and west, but there is some kind of
stokers' union; and the cattleman does not know whether he would be
welcomed among the stokers. There are always ways of getting across, but
the cattleman, or at any rate the young cattleman, needs to be posted up on
them.

Where did you stow away? asked one of the wizened partners of that
youth who morning by morning demanded his shaving water from Rafferty.
Michael had already begun his story, and this question, and others
discharged from the rear of those clustered near him, slightly offended him.
As I was saying, said he, I goes on board, and some of the fellows
had left one of the boats afore fixing the tarpaulin down. I gets inside there,
and I hears somebody say: 'I see you! I'll get the police to you!'
The Inquisitive One unconsciously ducked his head into his shoulders,
and the edges of his eyes narrowed. The word police always affected him
like that. Jack took on the expression of someone who does what is called
looking the other way. He became blank.
But I thought I knew the voice, continued Michael. I says: 'Is that
you, Jim Larson?'
It was a friend, was it? a tense listener exploded.
Michael looked with his one eye at the interrupter.
So he fastens up the tarpaulin, said he, and there I stays till we drops
Ireland, and I tell ye I was wantin' something to eat. So I puts my hand over
the gunwale, and loosens them ropes, and——
One of the men at this came a little closer, cunning and critical.
—I comes on deck, and oh there was a——
Michael's vocabulary broke down at this, and with a lot of by-thises and
by-thats, he gave it to be understood that a bo'sun and a third officer told
him that they would clap him in irons, that the skipper ordered him to be
swung over the side in a cradle and start in chipping, and that he said he
wouldn't go. At this point Michael looked up at the insidious critic.
Oh, well, indeed, he hurriedly went on, I did a bit of painting for
them, and my friend on board gives me a chance to slip the coppers at
Montreal.

Yes, said somebody, but wasn't there something about you having a
fight with the bo'sun when he took you forward?
There was indeed, there was some kind of scrimmage. Michael looked
up with his one eye at the man whose expression in listening was different
from that of all the other listeners. You been over with me before, haven't
ye? he asked him.
Tell us about the scrap you had with the bo'sun going forward! shouted
another.
No, indeed, Michael declared. I'll drop that bit out. I've told the story
so often that I don't know myself now which is the right way of it and
which is the wrong way.
There was a laugh at this, and Michael smiled.
Oh, indeed, there was a fight all right, he assured them. But I've told
about it different ways. I sometimes wonder myself now if I came off best.
There were sympathetic murmurs.
Indade, of course, of course, Mike spoke, lying stretched upon his top
bunk, near the door, head on hand, lenient and understanding. You got over
anyhow, and you didn't get put in the clink, and there's much to be thankful
for.
Oh, we're only cattlemen, said a voice.
Lend us your mouth organ! cried a youth.
The Inquisitive One looked for a moment as if he would protect his
breast pocket; but fighting was getting stale, and so he handed over the
instrument, the man who took it wiping it on a dirty sleeve before he
plunged into the strains of Rule Britannia! As he played there was a
movement among those near the door. Candlass was there, but he lingered
outside until the air was finished and then—Feed and water, boys, he said,
looking in, and as his men defiled into the passage Rafferty arrived.

Come on wid youse, lower deck! he bawled. His men filed out fairly
orderly. It was only at the morning call that they were still inclined to be
cross-grained.
Affairs were settling down into the routine of the trip. There was, indeed,
a spirit of friendliness growing among the Push. Free of liquor, and of the
after effects of liquor, the largeness of heart of many was evident, though
perhaps there was something morbid, as well as of kindly interest, in their
sympathy that they lavished on little Michael. He had his head turned over
it, spent most of his spare time sitting on the edge of his bunk, holding up
his head to let them look at his eye under the shade. Cockney and he, if they
had not yet made friends exactly, had allowed the matter of their fight on
the poop to be as an ancient matter now forgotten. The bad eye might have
been the result of an accident for all that was said about Cockney by those
who looked at it; indeed Cockney was the only one who seemed to recall
the origin of it. He sat apart, looking a little ashamed during these
examinations of the injured member; but his shame soon began to give way
to jealousy, for had he not a bandage on his head—had he not an interesting
ear that might be pried at? Yet, take it by and large, as seamen say, a feeling
of amicability came to the ship—that is to say by comparison with the spirit
that had inhabited it so far. Had any quietist been spirited aboard upon an
Arabian carpet he might well have been excused for stepping hastily on to it
again, and most hastily murmuring the incantations that would speed his
departure; but for those who had seen the Push with the drink in it, or the
drink waning in it, the S.S. Glory was now almost on the way to being
sacred!
The night-watchman, who slept away most of the day in Rafferty's cabin,
was the most objectionable sight on deck. He always appeared at meal
times, scooped up more than his share, then strolled about for a little while
for a constitutional, but was never spoken to. As the days wore on, however,
he spoke to others in a manner horribly blending intimidation and fawning,
his great moustache waving. He would plant himself in front of some
member of the Push and explain that he had come down in the world, that
he had a son in the Household Cavalry, six foot three, with a fist that would
fell an ox. If my son was on board, he would say, and glare, and if the
glare was returned: Oh, not that I mean anything, he would add. The

cattlemen gossiped infinitely less than do people aboard a passenger ship,
but it was inevitable that the watchman should be observed, and to some
extent discussed.
What was he saying to you? asked Jack of a young man before whom
the night-watchman had been peering and glaring and fawning.
Oh, I don't know—about a son in the army, six foot three, knock the
stuffing out of anybody. Says he's been divorced.
Mike, hanging over the rail, turned around.
He's a lazy good-for-nothing, that night-watchman, he said. It's a
wonder to me youse fellows on the lower deck don't fix him. These last
nights now we haven't had a dacent sleep for him waking Rafferty. He
laughed. I hear Rafferty says to him: 'Don't you waken me,' he says, 'if
there are only one or two loose. Waken me if there's more than half-a-
dozen.' Mike paused, and then added: But there always is half-a-dozen.
Some of the lower deck men within hearing grinned.
Oh, I know what it is, said Mike. Some of youse slips out at night and
loosens them, so as to get back on Rafferty for treating you the way he
does. It's cutting off your nose to spite your face, bringing Rafferty in at
twilve, at wan, at two, and at three, roaring like hill for you to tumble up,
and wakenin' us all. What was he after saying now, shoving his face at you,
me lad, and waving his tusks at you in the wind? Was it about his tall son
that has the strong arm?
He says he was divorced, said the young man.
Divorced, is it? answered Mike. He must have been married then, so
there wouldn't be any truth in what I would be calling his lad to him if he
comes along to me talking about him and his strong arm, and hinting what
he would be after doing, and him thousands of miles away. His voice
growled on. Did he tell ye what he was divorced for?
No.

Mike's voice almost suggested that he knew himself.
Indade, he was divorced for laziness, he said.
Jack swaggered away smiling, and the night-watchman, arriving then on
the poop, came up to him, seeing he was alone.
Are these men talking about me? he said.
He was evidently a poor judge of character. Jack strolled slowly past and
over his shoulder—Ask them, he said.
The night watchman glared and bellowed, in the roaring voice of a bar-
room bully: I'm only asking you a simple question.
Jack stopped in his stride, looked again over his shoulder, and smiled
queerly. The night-watchman thought it was a pacific smile, and stepped
closer.
I won't have it! he roared, and thrust his tusked face forward
presumably to let Jack see the determination in it.
Jack merely canted himself backwards, hands in pockets, and—Take
your face off me, he said quietly, or I'll spit in your eye.
The night-watchman was shocked.
That's a nice thing for a lad to say to an elderly man, he commented.
Oh, shut up! said Jack quietly.
If my son was here——
If your son was here, said Jack mockingly. I know all about him—
he's six foot three, isn't he?—I'd pound the stuffing out of him. One of the
family is enough to be going on with. If you come chumming round the
decks after me any more, I'll come along and stick you in the ribs to-night,
when you're down there supposed to be watching. I will. I don't want you to
come talking to me. You'll waken up with a knife in you. Now, that'll do!

and he strolled on, leaving the night-watchman with a face of terror, but
drawing himself erect, and twisting his moustache.
Jack walked the length of the deck and turned, but stepping a foot to one
side so that he walked back, in his slow march, direct upon the night-
watchman. As he walked he took his right hand from his pocket, clenched,
and walked swinging it. Get out of the way! he said. Shift! The
watchman moved on one side. Jack walked on, wheeled, marked where the
night-watchman stood now, and, both hands in pockets again, he trod the
deck back like a panther, straight toward him.
You're doing this on purpose! boomed the night-watchman, squaring
himself again.
Jack raised his handsome and evil face.
You come around talking to me, he said, you say any more to me and
I'll fix you all right. The night-watchman stepped aside, and when Jack
turned at the end of that walk the watchman was scuttling down the
companion way like a rabbit into a burrow.
Nobody congratulated Jack in words. He was a dark horse. He was one
of themselves, but except with Johnnie he was not a clubable young man.
Men like Cockney, men like Mike, never spoke to him, nor he to them.
Sometimes, in the morning, after the watering was over, if he met Scholar's
eye, he would give his head a little jerk to left and say: Hallo! He was of
those who, when others talked, could move away and not come back again,
and yet be called to account by no one for such contempt. He was of those
who, if spoken to, could lean up against the rail, cross-legged, turn and look
gently up and down the frame of the questioner, then move away, dumb.
Perhaps it was Jack, and his partner Johnnie with his feverish devilry, who
were at the bottom of an opinion that began to be current on the lower deck.
The lower deck men, it appeared, thought that the main deck men were
somewhat lacking in spirit. They managed to pass on their devilish
restlessness to one or two on the deck in question, and these, thus affected,
had the air of looking for trouble. A handy theme offered, and they fell to
grumbling over the fact that they were three men short.

Men short, did you say? inquired Mike.
Things short. Do you call them three things men?
The complaining voices subsided, but there were glances cast at Mike by
one or two that were intended to be read as: Who do you think you are?
I've had enough short-handed, broke out one of the less easily
extinguished.
But here the routine interfered. A hail came from forward, and the men
on the poop, and the men in the cabin below, had to file away to the
afternoon feeding. When the main deck bunch spread out with hay and
buckets, Candlass appeared, coming down the narrow alley to see that the
men did not overdo the belabouring of those steers near the end where the
hay was, great beasts whose main thought was to make a meal off the
armfuls of hay that went past them while the steers at the far end looked
down the alley and lowed vehemently; to see, also, that the mood of
laziness in the men did not triumph over the mood of determination and
prevent the steers at the far end from having a fair feed; to see also that all
hands had tumbled out. So far he had had no skulkers in his crowd, but he
was an experienced cattle boss. He moved along slowly, edging sideways
past each hay-laden man. All were busy; he had merely to look on. Then he
spoke.
Isn't there a man short? he asked.
Nobody answered.
Tom, he addressed one, do you know where that fellow with the
mouth organ is?
Isn't he here, boss? and the man that Candlass had spoken to looked
along the decks as if he expected to see the Inquisitive One somewhere at
work. Candlass went slowly up the alleyway. Scholar did not observe his
approach until the boss's hand was on his shoulder, and he pushed his
armful of hay aside to let Candlass go past, a steer on the side toward which
he moved immediately tearing at the bundle.

There's a man short, isn't there, Scholar? asked Candlass.
Don't know, answered Scholar, and was aware that Candlass peered
sharply at him before hailing Mike.
What's the matter with that man, Mike, the man that has the mouth
organ?
There was distress on Mike's forehead as he answered: I don't know.
You should know, said Candlass. You're the straw boss.
Yes, yes, I'm the straw boss maybe, but I'd rather work meself than
—— and he said no more. Only Scholar, near Candlass, caught the
response of: Oh yes, quite so.
Then the boss went aft; and all the men along the alley, for some reason,
turned and looked at his back. Even after he had disappeared they continued
to pass the hay without a word, then they looked along the alley again, and
coming forward was the Inquisitive One. The mouths of several of the men
opened, an upright furrow showed between their brows. What they saw
seemed inconceivable, for the Inquisitive One appeared to have shrunk, was
deathly white, did not look the same man. Behind him Candlass walked,
shoulders a little bent, as one under a burden, lips puckered, and eyes on the
deck; and the Inquisitive One fell to work, making a whimpering sound
ever and again. He was changed, as a cat that has been dipped in a tub of
water, but he never told any of the men what Candlass had done to him.
Some asked, who had his gift, or failing, of inquisitiveness; others left it to
him to tell if he cared to; but none heard. Probably it was a bear-hug that the
Inquisitive One had received, alone in the cattlemen's cabin where he
sulked over Mike's contempt for those who objected to working with three
men short—for Candlass had arms like steel.
CHAPTER XIII

The crew sober was very different indeed from the crew drunk. Their
likes and their dislikes were more explicable now. There were one or two
who spoke to nobody and were left alone, such as the Man with the Hat. He
had made a nest of hay for himself on the upper deck; nobody knew,
nobody cared, what he did when it rained; nobody was curious enough to
go along to see how he weathered it when they passed through lashing rain.
He had one manner for all men—one attitude—the attitude of a bulkhead. A
friendly approach was met by him exactly in the same fashion as an
inquisitive approach. As for openly antagonistic approach—none made it.
He did not seem to want to know anything about the cattlemen. Even when
at work with his half of the gang he was never known to say a word, except
once when a man pushed him, and he whirled round upon him and said, low
and vindictive, the one word Quit! And the man quit. The night-
watchman halted beside him once and said Good evening, but received no
reply. He did not take the snub, stood beside the nest of the Man with the
Hat, looking up at the voluminous and oily-looking smoke that rushed away
from the top of the smoke stack and stretched out like a fallen pillar,
diminishing across the sea.
Well, said the night-watchman, still looking overhead, it looks as if
we might have a dirty night.
Still there was no reply, and the night-watchman, thrusting his hands
deep in his coat pockets, fumbling for pipe and matches, looked round at
the Man with the Hat, and peered at him from under his cream-coloured
eyebrows—then moved on with a little more haste than he usually
exhibited, recovered a few paces away, and made pretence that he had only
moved off to light his pipe in the lee of one of the sheep-pens. He bent
down there in the attitude of a boy at leapfrog, and as he lit his pipe,
expending many matches, could only think to himself: That is a dangerous
young man.
Scholar, who had no distaste for the appearance of the Man with the Hat,
marching to and fro on the swinging deck later on, enjoying the pillar of
smoke rolling out in the deepening purple night, enjoying the wind,
enjoying the sweep of the masts that gave the stars, as they came out, an
appearance as of rushing up and down the sky, commented, in passing the

Man with the Hat: Bit of wind. No reply! He thought that the wind carried
his words away.
Bit of wind, I say, he repeated. No reply. He thought the man must be
deaf, so passed on and took his stand near the stern that tossed high and slid
down, every slide being a forward slide, the screws whirling. He was
enjoying the motion and the spindrift on his shoulders—for he was only in
undervest and trousers—when up came two men of the lower deck squad,
and one said to him: Rough night. He did not feel inclined to talk with
them, but, a little sore from what might have been a snub forward (for the
Man with the Hat might not be deaf), he put a certain warmth into his nod
and smile in response. The two came closer at that. He wondered why it
was that so many of these men could not chat without having the
appearance of being ready at any moment to lift a hand and smite their
interlocutor. They came close and plied him with questions—one a
Welshman, the other from the Kingdom of Fife. Somewhat thus went the
conversation:
Whit deck are you on?
The main deck.
I wondered. I never seen you on the lower deck. Where have you
been?
What do you mean? asked Scholar.
Have you been in Canada?
Yes—part of it.
What part? asked the Welshman.
Oh, I came up through Lower Ontario.
Then you wasn't stopping there? this from the Fifer, with a villainous
scowl, as if Scholar had been trying to deceive. You was in the States?

Instead of giving them County and State as reply, he answered now with
the bald: Yes.
What states? asked the Welshman.
Michigan.
Whit was ye daeing in Michigan? asked the Fifeman.
There came into Scholar's mind a brief conversation he had overheard
earlier in the day. One man had told a story of something he had seen when
I was in Florida.—What were you doing in Florida? the Inquisitive One
had asked after the story was told.—Eh? had said the man who had been
in Florida, with a note of warning.—I asked you what you were doing in
Florida? the Inquisitive One had returned, with a showing of the teeth.
—Ask my elbow! had been all the answer to that, spoken as if each word
was a knife-thrust. Scholar felt himself out of his sphere. He had no practice
in saying: Ask my elbow! in that tone, or in any tone; and it seemed to
him the requisite reply now. As he paused, wondering how to fob off these
two catechists, the Fifer said, with a curl of his lip: You're getting it now,
then.
Getting what? I don't understand you.
Oh, you understand all right.
Scholar's eyelids came slightly together. He wished he knew how to act
in this society, found himself squaring his chest a little, found that his jaw
was tightening. At this juncture Mike appeared on deck, hitched his belt,
came rolling along towards them, drew up alongside and yawned loudly,
stretching himself, raising his elbows in the air, and clasping his hands
behind his head. Then, leaning forward between the two catechists, he spat
out into the flying scud, turned his big back on them, hitched his belt again,
and said to Scholar: Bejabbers, it's cold! Let's have a quarter-deck walk,
Scholar.
Scholar fell in step with him. At the end of their walk, when they turned,
he was aware, without looking too keenly, that the two men of inquisitorial

mind were feeling highly vindictive; but the end of their return walk
bringing them again close to these two, Mike took a brief farther step to the
taffrail, swinging back largely.
What was them two saying to you? he asked, as they walked forward
once more.
Oh, just asking questions about where I had been, and all that sort of
thing.
Mike gave a Huh! of disgust. They wheeled, and began the return
balancing walk to the poop just in time to see them two going down the
companion-way. Mike brought up against the taffrail at the end of their
march this time, and leaning back on it, said he: I tell you what it is,
Scholar. Them fellers think they're better than us cattlemen. They're
tradesmen. I've seen enough—I don't need to listen to all they're saying,
after what you tell me. They're tradesmen; indade, I expect they're ruddy
plumbers. They've spotted you, you see. They're thinking to themselves:
'Here's a fellow on board here, and in the Ould Counthry we'd be putting
gas pipes in his father's house, and he's down now, and we'll kick him.' Just
the same way they would try to kick us too, if they didn't think we was
down already, beyant the likes of them to kick, he added in a grim tone, if
they didn't know that we knew how to fix them. If they come prying at ye
again, Scholar—listen now to what I'm tellin' ye: Turn yourself around
sideways to them, and says you to them, says you: 'Ask me elbow!' says
you. And if they shoves their face up against you, says you: 'I'll spit in your
eye if you shove your face at me like that!' And hit, Scholar, hit! It's
different with the likes of us. You came in among us like a man; anybody
could see you wasn't accustomed to us. Now you know what I mean—you
understand? said Mike, for he felt there was more in his mind than he
could express. I would rather go on a boat with you, Scholar, than with
thim, if it was a case of taking to the boats; and if it was a row on the
waterfronts I'd rather have you with your back to the wall with me than
them plumbers. You was born different, and you don't understand thim—ye
see what I mane, and he waved his hand. But you would niver roll a
shipmate; and if it came to the bit, I can see it in your eye, Scholar, you'd
hang on like a bulldog.

Scholar felt a great friendliness in his heart to this man, though he feared
he could not quickly learn the lesson, and would have to think out some
method of his own. The spit in your eye method of address was foreign to
him as yet. Mike had been shouting towards the end, for the wind was
rising; but now he paused a spell, and his gaze roved round the night and its
stars. He drew a deep breath and returned to matters mundane.
That watchman will have to keep his eyes open to-night, he said. He's
another of them. He frowned, looking along the decks forward. I wonder
if that feller wi' the big hat is along there yet—like a dead burrd in a nest.
He's blamed unsociable, that feller in the big hat, he commented. And then:
Oh, I don't blame him if he wants to be that way.
Perhaps he's afraid of being asked questions, suggested Scholar,
laughing.
Him! No, it's different with him. I said: 'Good evening mate,' to him the
other night there, and he pays no attintion. And I looks at him, and he gives
me the look—you know what I mane; so I says to him, says I: 'All right,
shipmate,' I says. 'All right, if that's the way of it. I know now, anyhow,'
says I to him, says I. He's a great lad, ye know. I was hearing about a bit of
a spar him and the cook had. He considered the darkened deck. Yes, he
could fix them two plumbers all right that was asking you questions.
Scholar had a certain depression in his heart. Mike was perhaps aware of
it.
Oh, I'd rather have you than him any day, all the same, said Mike, as if
in response to a spoken regret at inability to learn the ways of the society on
board. I think I'll turn in now. Remember what I was telling ye about them
gas-fitters.
Mike rattled down the companion-way, but Scholar remained on deck. A
faint sound of voices came from below, now and then a laugh. The decks
throbbed with the everlasting engine; a hissing and a scudding went along
the weather side; a sheep snuffled and bleated; a little while ago fresh
lashings had been put round their pens, tarpaulin dodgers protecting the
tops. There seemed to be nobody about; here and there a lozenge of golden

light, of deck lights, showed. The night was fallen almost as dark as the
smoke from the smoke-stack. The Glory tossed and slid, tossed and slid
onward; spray rattled with a sound like handfuls of shot on the tops of the
sheep-protecting tarpaulins. From forward the sea's assaults began to sound
more loudly, with many a resonant clap, and then the rattling as of grape
shot followed. Scholar thought he would go below, among his fellows.
Friendliness was very dear to him. It was only prying and worming into him
that ever caused his jaw to tighten, his eyes to narrow, as he wondered what
the stage directions might be.
CHAPTER XIV
When Scholar descended out of the tearing night he choked like an
asthmatical man. It was not now a smell as of fresh cattle that filled the
cattlemen's safe, called cabin; it was a suffocating smell as of ammonia.
Somebody was singing in the cabin that rose and fell with steely and
wooden screams, and with whispers of the sea running round it, the
tremendous sea that swirled and broke and sprayed on the other side of the
thin iron plates. The tobacco smoke was perhaps not quite so thick to-night,
for tobacco was growing scarce; but there were still plenty of pipes a-going
for blue clouds to temper the callous glare of the electric light.
Scholar slipped into the cabin, feeling for a moment almost shy. He had
learned how to come into the cabin when it was a kind of bedlam; but to
come into it now, and find it a kind of temperance sing-song hall for poor
seamen, with several of the poor seamen glancing at him in a way that
suggested their thought was: Ah! we'll ask him to sing next! was a little
upsetting. He tried to efface himself in his bunk. The applause following a
heartrending solo about For the flag he gave his young life! had just
ended.
Charles will give us a solo upon the mouth organ, said someone.

Charles looked bashful; it was one thing to play the mouth organ on the
dock front while the others double-shuffled (or, for that matter, to play it on
an ordinary evening when the ordinary life was going on, some listening,
others talking, voices roaring: You're a liar! others bellowing: Shut up!)
but quite another to have everybody quiet even before he began to play.
Charles screamed that he was fed up with the thing! and very likely felt a
qualm in his heart so soon as the words left his lips, for he was not at all
fed up with his mouth organ; he was very keen on it.
Many coaxed him, and one-eyed Michael said: Well, never mind if you
don't want to play. Don't worry the young fellow if he doesn't feel inclined.
Jimmy there will play.
Jimmy had been shouting: Go on, Charlie! For a moment he was like a
sailing ship taken aback, but he plucked up courage, and accepting the
instrument that Charles handed to him, wiped it with his sleeve and began
to play. Some rose and tried to dance, but did not find dancing easy, for the
gale was rising, and the stern rose and swung and fell and leapt up. They
danced, collided, and fell, danced again, and the onlookers whooped with
amusement, or smiled with mild disdain and pity; and the mouth organ
warbled, while the sea echoed and whispered round. Candlass, appearing
unexpectedly with a lamp, brought the man with the mouth organ to a stop,
and the dancers reeled to their bunks, where they sat down laughing.
Mike! said Candlass. Oh, you're there, Mike. Bring two or three of
the men forward with you.
Mike slipped over his bunk side; three or four others rolled out of their
own accord, the Inquisitive One among them, for though it must be a call to
work of some kind, and he was not eager for extra work, he simply must
know what was afoot.
That will do, said Candlass. I just want you to come along here and
see to some of these ropes before you turn in.
Away they went along the reeking decks. The cattle were not in a bad
plight at all; they had their four legs to stand upon, and propped each other
as well. It was those upon the lee side that gave most concern to Candlass

now. He carried the lamp high, casting weird shadows, and directing the
men in the slacking of a rope here, the hauling up of one yonder. There was
no doubt that the gale was rising; sailors were battening down a hatch
overhead, and their voices, as they hailed each other before they got the
whole hatch covered, shutting out the night, came down broken and blown.
Seas came over the decks, smacking like the flat of a great hand, and rushed
past. Now that the hatches were battened down there was a kind of confined
feeling—the long deck above, and the steer-packed deck below, converged
in the perspective, and gave a feeling as of being buried alive in a
monstrous box full of a dance of weird lights and shadows.
Their work over, Candlass said: That'll do, men. I'd better have a man
or two up to-night, along with the watchman.
All right, answered Mike, looking forward to the variety.
No, no—not you, said Candlass. You fellows can go back.
Away they went along the choking decks, one or another pausing now
and then to scratch, with closed fists—fingers being useless to the big
beasts—some head that thrust forward inviting. Others, when a head leant
out determinedly, smote at it to make way—but most, by this time, had
desisted from such methods, and were more inclined to make friends with
the steers. They met Rafferty as they were on their way back.
Where's Candlass? he asked them.
He's behind.
Oh, he's behind, is he? Are you fellows going to have another man or
two up with the watchman?
I was just talking about it, came Candlass's voice, he walking aft in the
rear; but I guess I'll stop up myself.
All right, said Rafferty. I'll relieve you then, if you tell me when.

They passed on to the cabin, Candlass following to thrust his head in and
look sharply till silence fell.
You fellows, he said, if I come and call on you to-night, turn out
lively.
All right, boss, several shouted, but Candlass had already turned away.
All were soon asleep; but, as it happened, there was no night call. On a
night like this, even if the bosses had not been about on the decks, the little
trick of loosening some steers in distaste for the night-watchman or for
Rafferty would have been allowed to lapse. All slept, or at least all were
silent; for perhaps here and there, in a bunk, someone lay staring at the
electric lights that were never put out, and could not be put out, there being
no switch in the cabin, lay staring and wondering at the whole business, the
deep breathing, the occasional sighs, the place ringing to the blows of the
sea, and echoing, as though someone whispered to the sweep of the spray
without; the whirl of the driving propeller going on and on, as if for ever,
under foot.
They thought at first, when they were called, that it was a night call,
woke gasping in the reek of ammonia, to find Candlass going his rounds
along a sloping deck, the Glory now having a tremendous list on, never
swinging up to a level, but rolling all the time from the degrees of that list
to a slope comparable with that of a church steeple, an almost anxious
slope, then back up again, and pitching, too. The men who were already
wakened began to shout: Tumble up! Tumble up! even before Rafferty
appeared; and there was little need for him to raise his cry, for almost all
were awake and rolling from their bunks as he lurched in at the door and
glared round. The wind shrieked outside, the cabin echoed more than ever
like a steel drum, the screams and groanings were infinitely louder.
Candlass looked at his men to see what fettle they were in, but he had
already arrived at an opinion and a computation regarding what men could
be relied on in the event of emergency.
Come on! said Mike, and led the way.

Scholar followed, Michael came next. It was very dark. They went along
on the windward side. All the cattle there had their broad fronts against the
making-fast board, their heads over it. The men moved along, propped
against the hoardings to leeward. The cattle on that side were standing well
back, leaning against each other, tails against the backboards. As they
manoeuvred forward a faint glow showed to starboard, which had nothing
to do with the scattered lamps that, from the beams above, swung round and
round in circles. One hatch (and only one) was still uncovered, and down
that the shrieking and roaring song of the gale came. Mike poised along
ahead like one walking on a steep roof. Up soared the Glory, and down she
plunged, and over she rolled—farther over, trembling down. The cattle
staggered; there was a sound of clicking horns, there were sounds of things
going slide and crash all over, and still she rolled. She had a list on her
beyond anything that Mike had ever known. He hung on with his right
hand; he was under the hatch now, Scholar a pace or two behind, and both
could look up at the dark sky overhead showing purple before the beginning
of another day. It was then four o'clock. And as she hung over thus they
watched the stars rush wildly up the sky like soaring rockets, up and over,
and then up came the sea, following the soaring stars. It gave them pause.
So far over did she hang that, from where they held tight upon the
windward side, they could see clean through the hatch above, and over its
lee edge, right out to the junction of sky and sea (a strip of awesome
whiteness, or less whiteness than the colourless look of a glass of water)
beyond an unforgettable tremendous tossing waste of a deep and velvety
purple. And still she hung over, so that they saw more and more of the sea.
It seemed to be rushing at them with all its great dark purple hollows, its
purple hillsides, its snowy crests. And in that moment Scholar averted his
eyes from it and looked toward Mike, and found Mike—hanging on—
looking over his shoulder rearward. Their eyes met. And Scholar believed
that perhaps Mike was right in his view of him that he had voiced the day
before when the gale was rising—believed that when it came to the bit he
would not be found wanting.

It seemed to be rushing at them with all its dark purple

hollows, its purple hillsides, its snowy crests.
Then the stars that had rushed up came rushing down again, bringing the
sky with them, and it fitted over in place. The Glory rolled and pitched
onward, still with something of a list, but no following roll sent her so far
over, and from no succeeding roll was she so slow to rise again.
CHAPTER XV
A few of the pickpocket-faced ones hung back during the gale that
morning, crawled into corners, effacing themselves, like sick cats. At the
afternoon feeding and watering (despite the words of contempt, glances of
contempt, and, worst of all, silences of contempt, bestowed upon them
when they showed face at their own feeding-time) several did not turn out,
pretended to do so—perhaps tried to do so—but slunk back to the cabin.
When Rafferty, missing them, came aft to hunt them forth, they showed
their peeked faces to him, worn and scared; and he despised them and left
them, turned back to his working majority again and shouted through the
shouting of the storm overhead, and the rushing of the draught along his
deck: There's some chickens, some chickens! His men knew to whom he
referred, looked at him—and sneered, and laughed, and tossed their heads
in agreement; Jack even, whose attitude to Rafferty so far had been one of
watchfulness, gave a kind of loud mutter of: We don't want them with us,
messing about here. Cockney too, energetic straw-boss, looked on them as
did Rafferty.
Let 'em lie there and shiver, then, he said.
Only two of the main deck men were perturbed beyond labour by the
steadily increasing violence of the gale, scared by the consideration that it
had begun to blow last night, and had been getting worse and worse ever
since.

Two men short! commented Candlass in the afternoon, and went aft to
the cabin to look for them, found one on the way, behind a bale of hay,
peered at him as if wondering what he was doing there, balancing carefully
with loose knees, taking hand from pocket only to grab and hang on by a
protruding end of barricade. He eyed him as a man may eye a newly-bought
puppy that has gone in between the sofa's end and the wall. The youth got
up, scrambled out as best he could, hauled himself to his feet. Candlass
spoke never a word, but bowed to him in the attitude of one listening for a
whisper, mock-commiserating, and the youth dragged himself forward to
find that his fellows did not want him, had fallen to work passing the hay
themselves, and were inclined to treat him as if he was in the way. He had
the air as of pleading to be allowed to do something. Candlass, meanwhile,
walked on into the cabin, zig-zagged across, looking for his other missing
man.
There were two of the lower deck hobbledehoys there. He waggled a
thumb at the door, and they got up and crawled out, but he did not follow
them; he went up on deck instead, to hunt out the man who was missing
from his own deck in particular. The sheep sniffled and bleated occasionally
under securely-lashed dodgers that now covered the tops of all the pens.
They saw his feet and thrust out their black faces, wrinkled their noses,
shivered and withdrew. It was near their feed time. (Mike and Cockney,
with two or three others, saw to them daily on their way back, after having
tended the cattle.) Candlass tilted his body along, looking left and right to
see where his man might be hiding, the ship ever and again pausing in the
midst of a rise, pausing much as men on deck did at a more violent and
unexpected roll and kick. Some greater wave, at such times, had caught her
fair, and smashing upon her hull as on a cliff, raced whirling along the
length of her, shot up her side, soared thinly there beyond the bulwark, to be
immediately blown wide, as is the top of a fountain in the wind, scudding
and rattling along the decks. Tarpaulins had been rigged entirely across her,
below the bridge, to protect the sheep on the after deck; and as far as to that
barrier did Candlass now strut, tilting and balancing. And there, in a space
between two sheep-pens, beside a ventilator, he saw a pair of boot-soles,
bent down and grabbed at the legs beyond them, and the face of the missing
man looked up at him—green. It was sea-sickness. Candlass stooped low.

Sick? he said.
The man's eyes rolled. He clung desperately to the ventilator.
Don't fall overboard, don't want to lose a man. Savvey?
The man tried to nod; his whole body sagged forward in that effort.
You lie on your back when you ain't actually being sick, Candlass
roared into his ear. Savvey?
Again the man tried to nod and at least succeeded in making his head go
up and down instead of being powerless to keep it from doing aught but
rolling left and right.
Don't fall overboard, Candlass counselled again, and lurched away,
muttering to himself: Sick all right.
But most of the men enjoyed the gale. It was something doing. And
when, next morning, the pickpocket-faced youth sat up ready to give his
shout of: Call me in another hour, Rafferty, and bring me me shaving
water, his voice failed. He looked round the cabin; he had been one of the
shirkers yesterday.
That's right, said one of the men. You keep your mouth shut this
morning! And the gunsel and his special cronies kept quiet, for it was
unfitting that those who skulked in a corner during a gale should cheek
Rafferty the morning after merely because they found that the swing and
sweep of the tossing stern were back a little more to the normal. The gale
had indeed blown itself out, or nearly so. It was a tremendous morning in
the North Atlantic. A fountain of gold, preceding sunrise, shot up eastwards.
A sound of hissing and of breaking foam was round the ship, and echoed in
every corner. The waves soared, great and curving, blue and purple, veined
like marble in their forward curves with the foam of other broken waves,
soared higher, curled their tops, broke, and as they broke the wind took the
foam and whirled it broadcast. There was a wonderful purple and blue and
windy hilarity over that great expanse, so high a sea running that even the
horizon line was ragged.

The grub that day seemed painfully scanty. The uneaten shares of the
one or two seasick men made no difference, so great were the appetites of
the fit. Cockney admitted, after the meal was over, that he sympathised with
those persons who chummed with, or intimidated, Pierre and Four Eyes, for
the sake of what food they might smuggle away from the galley—though
his phrasing of this comprehension was of course all his own.
Are they cadgin' off Frenchy and that object in the coat? asked Mike.
'Aven't you seen 'em? said Cockney. 'O, Frenchy, bring hus along
some pie!' he cried out in a fleering voice.
I quite belave it, said Mike. I see some of 'em cadgin' tobacco. There's
men aboard this ship I know I wouldn't prisint me plug of tobacco to. If
they took a bite out of it you'd be thinkin' the plug was in their mouth, and
the chaw they axed for was the piece they gave back to ye. There was an
attempt at a laugh, an obvious attempt, for the shot had gone home. 'Have
ye got a piece of chewing on ye?' mocked Mike. 'Me pipe's empty, have
you a fill about ye?'—'Have ye a ceegareet about ye?' He paused. 'After
you wid the ceegareet!' he mocked.
And he lived up to his opinion. There are people who arrange their moral
code according to what they can do and cannot do. There are people to
whom a fall from fealty to their code is occasion for renouncing and
deriding that code. Mike was not of these. He disliked cadging, but had his
love of a smoke or a chew driven him to cadge he would not have
relinquished his opinion; he would have smoked and chewed as a defeated
man.
All day now there was sign of better weather. Even the wind aided to
calm the seas, swinging round a point or two and besoming the wave-tops,
flattening them. There was hash and pea-soup that day—the pea-soup drunk
in the tin mugs, of course, along with, or after, the hash—and the Push
were glad of it. It was a great tonic day. By night all the clouds seemed to
have been blown away; stars by the billion filled the vault; the Milky Way
was like a whirl of triumph, like a gesture of joy across the heavens. The
wake of the tiny little Glory (she seemed tiny now) was as an imitation of
that Milky Way, full of balls, large and small, and smaller, down to the size

of sparks even, of phosphorus—dancing and bursting and thinning out.
Mike, coming on deck a trifle disgusted by a surfeit of what he called
soup-kitchen palaver that was in progress amid a group of youse,
looked down at that wake, moody, and furrowed, that kind of half-broken
look upon his face, like a wondering beast, a puzzled beast. He stood there
at the stern, lifted high and brought low, till his back went cold.
Bejabbers, it's all very strange, he said to himself; and being cold he
looked round for shelter. Some wisps of hay, blown from windward, had
been brought up against the lee rail, and he gathered them together. The
sheep bleated.
I'm using this for me own comfort, he said, addressing the sheep in the
end cote; go to sleep! And he squatted down with his back against the
cotes, and stretched out his legs—sat there a long time while the ship pulsed
and pulsed on, tossing her stern and the engines racing and steadied, racing
and steadied, as she slid through the sea, churning the water into foam, in
which whirls of gold began like nebulas of stars, whirled into complete little
globes, danced away as entrancing as opals, and then suddenly went out.
Now it happened that, below, Scholar felt he might almost suffocate, and
remembering that he had been some time out of the weather, for which he
had always a great friendliness, never liking to be too long out of touch with
it—blow high, blow low, rain, mist or sunshine—he too came on deck. The
poop companion-way had been closed these last few days, and that made
the cabin all the more asphyxiating. He came up that narrow staircase, feet
clattering on the worn brass edges, turned the handle; eddies of wind did the
rest. He wrestled a spell with the door, then came on deck, closed the door,
and looked up in awe at all these stars—stood there balancing, now drawn
away from them down and down, next moment soaring and swinging up
with a sensation as if he might be swung on and come up through that
golden dust and see some explanation. Then down he was borne again, or
felt as though his body was borne down and his spirit left up there.
Explanation, or no explanation, it was good—all good, the crying of the
sea, the whistle and shriek of the wind in the cordage, the feel of the wind,
the scud of the spray—good!

He turned and looked forward. There seemed to be not a soul on deck. It
was as if he had dropped from a star, forgetting all about it on the way, and
had alighted gently upon this thing that, reeking volcano-like, tossed and
swung, but always forward through the night. He had almost to take it on
faith that there was a man in that hardly-discernible little barrel on the
foremast, the summit of which raked from left to right. He peered up at the
bridge. Yes, something moved there from port to starboard and back again,
like a mouse running to and fro on a shelf. Below his feet the ceaseless
whirl and whirl went on. A man suddenly appeared, jumping up on top of
the sheep pens, tapping with his toe before him, then stepping, to be sure he
stood on firm board top and not on tarpaulin cover, turned the top of a
ventilator, disappeared, bobbed up again, revealed against the starry sky, or
at any rate revealed from his head down to about his knees, the wind pluck-
pluck-plucking at his short jacket. He disappeared again, jumping down and
was gone. Scholar moved to one side, kicked something soft, looked down
and said: Oh, I beg your pardon! and a coarse Irish voice answered: All
right, Scholar.
There was fresh movement at Scholar's feet.
I seen ye against the stars, but ye couldn't see me. Bring yourself to an
anchor here beside me—I have some straw here—and give us your crack.
Scholar, peering down, was now able to make out where Mike reclined,
and sat down beside him, back against the end of the last sheep-pen. But
they did not speak at once. Scholar felt in his pocket for pipe and tobacco,
and held the tobacco-bag to Mike.
Have a fill? he said.
Mike put forth a hand, and drew it back.
No, he growled.
I've a plug of chewing-tobacco somewhere, said Scholar. Yes—here it
is.

Out went Mike's hand, then abruptly back again; and this time he thrust
both hands deep in pockets.
No, thank you, Scholar.
Scholar wondered if he had given some offence. Ignorant of how to repel
in this society in which he found himself, he might also, even in sitting
down in response to Mike's invitation, ignorantly have transgressed some
usage of courtesy in this sphere. Next moment Mike explained.
When I see the way the fellers on this ship go cadgin' for tobacco it
gives me a pain. He shifted his position slightly, as if he really felt a
physical pain. I would think shame to keep on axing a man day after day—
many times a day—'Have you got any chewing? Have you got any
smoking?'
That's all right, said Scholar. You didn't ask me—I offered to you.
Yes, yes, I know; but I said to meself: 'Thim fellers has no daycency. I'll
do without chewings and smokings until I get to Liverpool.' No, Scholar,
thank you kindly—I'll go wanting it. It has too much hold upon me as it is.
Scholar did not press.
CHAPTER XVI
Now there began to be signs of how the cattlemen would wander off
together when they came to land again. Understandings seemed to be
arrived at between threes and fours and half-dozens. It was not exactly
cliquishness—it was more a case of birds of a feather—No, that simile is
bad, as are most ready-made proverbs. Not their outward parts, their mere
feathers, but their inner parts arranged the groupings. The snarling was all
over; drink, and the effects of drink, were old stories. One or two men, of

course, were still left alone by all, men so different as the Man with the Hat
and the Man with the Specs. Frenchy, or Pierre, his tobacco nearly done,
and his complaisance in giving it away in a like state, was now discarded by
some of the former spongers, but not by all. Probably those who had been
interested in him, as well as sponging upon him, were the ones who now
besought him to sing a French song, or to tell them what France looked like.
The feeding and watering were by this time matters of routine, wakening
at four a habit. The cabin was almost tenantless, only the cold-blooded, or
those children of the slums who felt out of their element unless they slept in
rancid air, turned in there. Among the diminishing hay near the hatches—all
open again—or on the upper deck, around the smoke-stack, and between
the sheep-pens, most of the men slept, snatching a nap during the day when
the cattle did not call them, sleeping there at night until only the extreme
cold drove them down, with short gasps, from the windy deck to the
asthmatical cabin. It was, indeed, easier to tolerate the cabin by day than by
late night, for by day, and early in the morning, there was some tobacco
smoke—not much now, to be sure—and the companion was open. At night
the tobacco smoke soon ceased to combat with the ammonia fumes as the
men slept, and some of the cold-blooded were sure to mount up and shut the
companion-door before turning in, making the cabin's atmosphere more
stifling still.
They began to talk of reaching Liverpool, of what they would do there,
to ask each other: You coming back on her? Cockney and Michael
exchanged friendly speech again. It is doubtful which started, but they were
again conversing. The Inquisitive One begged Frenchy to come with us,
indicating the group round him; but Pierre explained that he was going
home. One told another about the loss of Frenchy's valise, and Mike's
recovery of it, as he might tell of the incident on another ship one day if
Frenchmen, or valises, were mentioned. Many of the men fell to rubbing
their chins, and announcing that they would be the better of a shave. They
asked each other: Have you a razor? Frenchy taking warning by the
cadging of tobacco that left him smokeless now, pretended that he didn't
know what razor meant, was unusually dense to signs, could not be got to
understand of what they talked. Somebody commented that he must have a
shave, that they all should shave, looked too tough, that the day after to-

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