SALMONELLA-INDUCED SYSTEMIC ACQUIRED RESISTANCE IN TOMATO AND ITS IMPACT ...

94 Pages·2015·1.7 MB·English

ABSTRACT

Title of Document: SALMONELLA-INDUCED SYSTEMIC
ACQUIRED RESISTANCE IN TOMATO AND
ITS IMPACT ON SALMONELLA
COLONIZATION OF TOMATO LEAVES

Tommy Phannareth
M. S. 2015

Directed By: Dr. Shirley A. Micallef
Plant Science and Landscape Architecture
Center for Food Safety and Security Systems
Salmonella enterica is an enteric human pathogen that lives in gastrointestinal
tract; however, Salmonella are able to survive in plants. Thus, vegetables such as tomato
are vectors for Salmonella. Evidence suggests that Salmonella induces PAMP-triggered
immunity (PTI) in plants, however, plant systemic acquired resistance (SAR), which may
act to suppress Salmonella populations, has not been explored. This research investigates
whether Salmonella triggers SAR in tomato, and whether SAR activation restricts
epiphytic Salmonella populations. Inoculation of tomato leaves with Salmonella
increased SAR marker gene expression in distal tomato leaves, but did not reduce
populations of the phytopathogen Pseudomonas syringae or Salmonella on distal leaves,
even following treatment with chemical SAR activators. NahG plants, which are deficient
in SAR signaling, supported higher Salmonella populations, and nitric oxide depletion on
leaf surfaces favored Salmonella growth, suggesting that SAR is involved. SAR alone is
insufficient to restrict Salmonella growth on tomato, despite being triggered.
THE INTERACTION BETWEEN TOMATO SYSTEMIC ACQUIRED RESISTANCE
AND SALMONELLA ENTERICA

By

Tommy Phannareth

Thesis submitted to the Faculty of the Graduate School of the
University of Maryland, College Park, in partial fulfillment
of the requirements for the degree of
Master of Science
2015

Advisory Committee:
Dr. Shirley A. Micallef, Chair
Dr. Wendy Peer

Dr. Shunyuan Xiao
© Copyright by
Tommy Phannareth
2015
Acknowledgements
To begin, I would like to express my deepest gratitude for the guidance I have
received from Dr. Shirley A. Micallef throughout my graduate career. In addition to the
invaluable support and patience she offered through the course of this thesis, she also
provided me excellent mentorship as I assisted her in teaching her course “How Safe is
Your Salad: The Microbiological Safety of Fresh Produce”.
Thank you also to Dr. Wendy Peer and Dr. Shunyuan Xiao for all of their help.
Both provided me with information and ideas that shaped the course of my research. I
would like to particularly acknowledge Dr. Peer for her guidance on experimental
protocols, and Dr. Xiao for his instruction during his Plant-Microbe Associations class
and for providing me with the Pseudomonas syringae pv. tomato DC3000 that I used in
my experiments.
I would also like to thank the undergraduate researchers who assisted me with this
project: Adriana Echalar, Marie Pham, Nazleen Khan, and Seun Agbaje. Seun in
particular assisted me throughout the summer of 2014, and mentoring him on
experimental design and aspects of microbial and plant biology was a joy and a pleasure.
I would also like to acknowledge the contributions of my fellow lab members
Sanghyun Han, Sarah Allard, Neiunna Reed-Jones, Rachel McEgan, Angela Ferelli, and
Louisa Martinez. Each of them provided advice, motivation, and additional perspectives
on my work. Additionally, Sanghyun shared with me tomato seeds and bacterial strains,
and much of my work was informed by his research.
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Table of Contents
Acknowledgements ........................................................................................................... ii
Table of Contents ............................................................................................................. iii
List of Figures .................................................................................................................... v
List of Tables ................................................................................................................... vii
Table of Abbreviations .................................................................................................... ix
Chapter 1: Introduction ................................................................................................... 1
1.1 Background and Literature Review........................................................................... 1
1.1.1 Salmonellosis outbreaks associated with fresh produce ..................................... 1
1.1.2 Salmonella adaptations for plant colonization.................................................... 1
1.1.3 The tomato phyllosphere as a habitat for Salmonella colonization .................... 3
1.1.4 Plant pathogen-defense mechanisms .................................................................. 4
1.1.5 Chemical activators of SAR ............................................................................. 11
1.1.5 Plant defense and Salmonella interactions ....................................................... 15
1.2 Rationale and Significance ...................................................................................... 17
1.3 Hypotheses and objectives ...................................................................................... 19
1.3.1 Salmonella triggers PTI which may subsequently induce SAR ....................... 19
1.3.2 Salmonella survival is hindered by tomato plants in which SAR has been
triggered. .................................................................................................................... 20
Chapter 2: Assessing the Hypersensitive Response and Systemic Acquired
Resistance in tomato in response to Salmonella ........................................................... 22
2.1 Introduction ............................................................................................................. 22
2.2 Materials and methods ............................................................................................ 24
2.2.1 Plant material .................................................................................................... 24
2.2.2 Bacterial strains ................................................................................................ 25
2.2.3 Salmonella multiple inoculation assay ............................................................. 25
2.2.4 Pre-inoculation assay ........................................................................................ 26
2.2.5 cPTIO Treatment .............................................................................................. 27
2.2.6 3, 3'-diaminobenzidine (DAB) Stain for ROS .................................................. 27
2.2.7 Statistical Analysis ........................................................................................... 28
2.3 Results ..................................................................................................................... 28
2.3.1 Salmonella influence on Pst survival on distal leaves ...................................... 28
2.3.2 Salmonella influence on Salmonella survival on distal leaves ......................... 30
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2.3.3 Influence of salicylic acid on Salmonella survival ........................................... 31
2.3.4 Influence of nitric oxide on Salmonella survival .............................................. 31
2.3.5 Salmonella and reactive oxygen species .......................................................... 32
2.4 Discussion ............................................................................................................... 36
Chapter 3: Influence of SAR activation on Salmonella colonization of tomato ........ 39
3.1 Introduction ............................................................................................................. 39
3.2 Materials and Methods ............................................................................................ 41
3.2.1 Plant Material ................................................................................................... 41
3.2.2 Bacterial strains ................................................................................................ 41
3.2.3 Bacterial inoculum preparation ........................................................................ 42
3.2.4 Bacterial Retrieval ............................................................................................ 42
3.2.5 Acibenzolar-s-methyl treatment ....................................................................... 42
3.2.6 Hexanoic acid treatment ................................................................................... 43
3.2.7 B-aminobutyric acid treatment ......................................................................... 43
3.2.8 Analysis of gene expression by quantitative reverse transcriptase-polymerase
chain reaction (qRT-PCR) ......................................................................................... 44
3.2.9 Statistical Analysis ........................................................................................... 46
3.3 Results ..................................................................................................................... 46
3.3.1 Chemical SAR activators and Pst survival ....................................................... 46
3.3.2 Chemical SAR activators and Salmonella survival .......................................... 48
3.3.3 SAR-related Gene Expression on Distal Tissues ............................................. 49
3.4 Discussion ............................................................................................................... 53
Chapter 4: Summary of conclusions, reflections, and future research ...................... 58
4.1 Summary of main conclusions ................................................................................ 58
4.2 Summary of additional conclusions ........................................................................ 58
4.3 Reflections and future directions............................................................................. 59
4.3.1 Reflections on experimental methods and materials ........................................ 59
4.3.2 Future directions ............................................................................................... 62
Appendix 1: Bacterial Counts ........................................................................................ 63
Appendix 2: qPCR Data ................................................................................................. 69
References ........................................................................................................................ 78

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List of Figures
Figure 1.1 SAR circuitry involving a network of signaling molecules. (Shah and Zeier,
2013) ................................................................................................................................... 7
Figure 1.2 Boolean representation of SAR, PR1, and PR5 network. Filled circles indicate
signal branching points. Bullets with flat left sides indicate “and” gates and require both
inputs, bullets with concave left sides indicate “or” gates, which trigger with a single
input. (Zhang and Shapiro, 2002) ....................................................................................... 9
Figure 1.3 Simplified model illustrating NO-ROS signaling in SAR (Wang et al., 2014)
........................................................................................................................................... 11
Figure 2.1 Pseudomonas syringae pv.Tomato counts three days post inoculation on
tomato cv.‘Primo Red’ plants pre-treated with bacterial suspensions. No significant
difference was detected between treatments by one-way ANOVA. Error bars indicate one
standard error. Results are pooled from two independent experiments, each with four
biological replicates per treatment. ................................................................................... 29
Figure 2.2 Decline Salmonella Newport counts on Tomato cv.‘Heinz’ and ‘Primo Red’
after 24 hours. No significant difference between treatments using Student’s t-test were
observed. Error bars indicate one standard error. ‘Heinz’ results are pooled from two
independent experiments with four biological replicates per treatment. ‘Primo Red’
results are from one experiment with four biological replicates, which was repeated with
equivalent results. ............................................................................................................. 30
Figure 2.3 Mean Salmonella Newport counts three days post inoculation on tomato
cv.‘Moneymaker’ or a NahG mutant of ‘Moneymaker’. The NahG mutants supported
higher levels of SeN colonization compared to the wild type (p = 0.04) .......................... 31
Figure 2.4 Salmonella Newport counts three days post inoculation on tomato cv.‘Primo
Red’ after spray treatment with H O (control) or cPTIO. The asterisk denotes a weakly
2
significant difference from the control (p = 0.105). Error bars indicate one standard error.
Results are pooled from two independent experiments, each with four biological
replicates per treatment. .................................................................................................... 32
Figure 2.5 DAB staining of tomato cv. ‘Primo Red’ leaflets 6 hpi. Results of two
experiments, split by row (n = 4, n = 5). ........................................................................... 34
Figure 2.6 Mosaic plot of DAB stain scoring of tomato cv. ‘Primo Red’ leaflets. Pooled
results of two experiments (n = 4, n = 5) .......................................................................... 35
Figure 3.1 Pseudomonas syringae pv.tomato counts three days post inoculation on tomato
cv.‘Primo Red’ plants treated with chemical SAR activators. Each treatment was
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significantly different from its associated control when compared with Student’s t-test, p
< 0.05. ............................................................................................................................... 47
Figure 3.3 Salmonella Newport counts three days post inoculation on three tomato
cultivars after treatment with chemical SAR activators. ‘Primo Red’ mock treatment bar
representative of independent ASM and HA experiments. Error bars indicate one
standard error. ................................................................................................................... 49

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List of Tables
Table 1.1 “ASM Activity in Important Crop Plants Against Various Classes of Pathogen”
Adapted from (Oostendorp et al., 2001). “+” indicates pathogen resistance. ................... 12
Table 2.1: Salmonella Multiple Inoculation Schedule. ..................................................... 26
Table 3.2 qRT-PCR Primers ............................................................................................. 45
Table 3.3 qRT-PCR conditions ......................................................................................... 46
Table 3.3. PR1 expression relative to the mock inoculum for each chemical treatment.
Rows with the same letter within each treatment are not significantly different from each
other when comparing ΔΔCt by one-way ANOVA with Tukey’s HSD post hoc test (p <
0.05). ................................................................................................................................. 51
Table 3.4 PR5 expression relative to the mock inoculum for each chemical treatment.
Rows marked with an asterisk failed to amplify PR5, and ΔcT was calculated with Ct
gene
= 35. Rows with the same letter within each treatment are not significantly
of interest
different from each other when comparing ΔΔCt by one-way ANOVA with Tukey’s
HSD post hoc test.............................................................................................................. 52
Table 3.5 SAMT expression relative to the mock inoculum for each treatment. Rows with
the same letter within each treatment are not significantly different from each other when
comparing ΔΔCt by one-way ANOVA with Tukey’s HSD post hoc test. ....................... 53
Table 3.6 Gene expression as a relative quantity to plants treated with a mock inoculum
(PBS) and a mock activation treatment (H O). Statistics performed on raw ΔΔCt values.
2
PR5 expression undetectable in the mock inoculum, mock treatment reference control, Ct
value imputed to 35. Statistics performed on raw ΔΔCt values. Difference letters denote
expression levels significantly different from each other within each gene. .................... 53
Table A1 – Pst counts on pre-inoculated tomato cv. ‘Primo Red’ ................................... 63
Table A2–SeN counts on tomato cv. ‘Primo Red’folowing single or multiple inoculations
with SeN ............................................................................................................................ 64
Table A3 – SeN counts on tomato cv. ‘Moneymaker’ or NahG mutants in the
‘Moneymaker’ background. .............................................................................................. 65
Table A4 – SeN counts on tomato cv. ‘Primo Red’ treated with cPTIO .......................... 65
Table A5 – Pst counts on tomato cv. ‘Primo Red’ treated with chemical SAR activators
........................................................................................................................................... 66
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Table A6 – SeN counts on tomato cv. ‘Primo Red’ treated with chemical SAR activators
........................................................................................................................................... 67
Table A7 – SeN counts on tomato cv. ‘Heinz’ and ‘Nyagous’ treated with chemical SAR
activators ........................................................................................................................... 68
Table A8 – SAMT ΔΔCt values ....................................................................................... 71
Table A9 – PR1 ΔΔCt values ........................................................................................... 74
Table A10 – PR5 ΔΔCt values.......................................................................................... 77

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