myrtle rust

Screening two wild-sourced Australian Myrtaceae for responses to Austropuccinia psidii (myrtle rust) and determining the underlying genetic basis to resistance

Peri Ann Tobias

School of Life and Environmental Sciences Faculty of Science The University of Sydney

A thesis submitted in fulfillment of requirements for the degree of Doctor of Philosophy

March 2017 1

Statement of Originality

This is to certify that to the best of my knowledge; the content of this thesis is my own work. This thesis has not been submitted for any degree or other purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged.

Name:

Peri Tobias

Principal supervisor:

Prof. Robert Park

Auxiliary supervisors:

Prof. David Guest and Dr Carsten Külheim i

Acknowledgements I am extremely grateful for the generous support and encouragement from my supervisors Professor Robert Park, Professor David Guest, of the University of Sydney, and Dr Carsten Külheim, of the Australian National University. I have also received support from a number of academic researchers and technicians at the University of Sydney, Plant Breeding Institute and at the Faculty of Agriculture and Environment. I would like to acknowledge the University of Sydney HPC service for providing resources that have contributed to the research results reported within this thesis. I would also like to acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the University of Sydney. Thank you to Digby Growns, Senior Plant Breeder, Kings Park, Perth, for generously assisting with locating Chamelaucium uncinatum populations. I am especially grateful for the love, encouragement and support of my husband, Mark Powrie, who took a deep interest in my research, helped me with my ventures into running software on the command line and assisted with python scripts that enabled searches within sequence data lists. I wish to acknowledge and thank my two sons, Axel and Jasper Powrie, who have always shown an interest in my work and asked probing questions that forced me to think about my results. I wish to thank the Faculty of Agriculture fellow PhD students for the support network provided around the lunch table. Lastly, I am grateful for the Australian Post-Graduate Award scholarship as well as research and travel funds from the Australian Government Rural Industries Research and Development Corporation (RIRDC). Top up scholarships were also generously provided by RIRDC and the University of Sydney.

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Publications arising from the research

2017

Peri Tobias, Nanette Christie, Sanushka Naidoo, David Guest, Carsten Külheim: ‘Identification of the Eucalyptus grandis chitinase gene family and expression characterization under different biotic stress challenges’. Tree Physiology DOI:10.1093/treephys/tpx010

2016

Nanette Christie,

1

Peri Tobias, Sanushka Naidoo, Carsten Külheim: The

Eucalyptus grandis NBS-LRR Gene Family: Physical Clustering and Expression Hotspots.

Frontiers

in

Plant

Science

2016

Jan

12;6:1238.

DOI:

10.3389/fpls.2015.01238, 1shared first authorship 2015

Peri A Tobias, David I Guest, Carsten Külheim, Ji-Fan Hsieh, Robert F Park: A curious case of resistance to a new encounter pathogen: Myrtle rust in Australia. Molecular Plant Pathology 11/2015; DOI:10.1111/mpp.12331

2015

Peri Tobias, Robert Park, C Külheim, David Guest: Wild-sourced Chamelaucium uncinatum have no resistance to Austropuccinia psidii (myrtle rust). Australasian Plant Disease Notes 04/2015; 10(1). DOI:10.1007/s13314-015-0167-0

Manuscript submitted 2017

Peri Tobias, David Guest, Carsten Külheim, Robert Park: De novo transcriptome study identifies candidate genes involved in resistance to Austropuccinia psidii (myrtle rust) in Syzygium luehmannnii (Riberry). Molecular Plant-Microbe Interactions

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Summary of the research Austropuccinia psidii is a biotrophic fungal pathogen, first described in Brazil, and the causal agent of rust infections commonly named myrtle rust. A. psidii was initially detected in Australia in April 2010 in a commercial nursery, and by late that year had become established on a wide range of species within the Australian Myrtaceae and was deemed ineradicable. Currently, it is believed that only one genotype of the pathogen is present in Australia, based on microsatellite markers which match an Hawaiian pathotype. Within Australia infection responses vary from complete susceptibility to resistance within a number of distantly related species, within populations and provenances, indicating a large degree of host genetic diversity. Previous research has identified a locus for resistance within Eucalyptus grandis indicating specific resistance may be present within some Myrtaceae. While there are concerns for Australian natural vegetation communities, dominated by Myrtaceae, established and emerging commercial species are also likely to be impacted. The following research takes two commercially important Australian Myrtaceae; Chamelaucium uncinatum, grown for the cut flower market and Syzygium luehmannii, an increasingly valuable berry crop, and examines their responses to inoculation with A. psidii. A brief summary from each chapter follows. Chapter One: A curious case of resistance to a new encounter pathogen: the Australian myrtle rust example This chapter is published as a micro-review in the journal Molecular Plant Pathology that reviews the literature related to host responses to new encounter pathogens and suggests mechanisms for observed resistance in Australian Myrtaceae. Using the myrtle rust case study, it examines models to account for the presence of resistance to new encounter pathogens, such as

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the retention of ancient resistance genes through prolonged ‘trench warfare’, pairing of resistance gene products and the guarding of host integrity. The manuscript is available in the thesis Appendices (Appendix 1). Minor amendments to the manuscript have been included within this chapter. Chapter Two: Screening two wild-sourced Australian myrtacaeous species for resistance to Austropuccinia psidii (myrtle rust) Two wild-sourced Australian species, with commercial value, were screened for resistance to Austropuccinia psidii infection. Chamelaucium uncinatum, Geraldton waxflower, is a West Australian endemic shrub that is grown as a garden ornamental and for the cut flower market. Syzygium luehmannii, Riberry, is endemic to the east coast and is becoming an increasingly valuable berry crop. Pathogenicity tests showed that wild-sourced C. uncinatum plants have no apparent natural resistance to this pathogen, while S. luehmannii shows promising results with 77 out of 103 (75%) of the genotypes tested presenting macroscopically visible resistance. Of the resistant genotypes, specific resistance in the form of localised necrosis (typical of a hypersensitive response) was determined in 29% of wild-sourced individuals. A manuscript regarding the susceptibility of C. uncinatum to A. psidii was published as a result of this research (Appendix 2). Chapter Three: Identification of genes involved in resistance to Austropuccinia psidii (myrtle rust) in Syzygium luehmannii (Riberry) To understand the molecular basis for Syzygium luehmannii (Riberry) responses to inoculation with Austropuccinia psidii, RNA from leaf samples taken at 0, 24 and 48 hours post inoculation (hpi) from both resistant and susceptible plants was extracted and sequenced. De novo transcriptomes for each plant, from all samples (pre-inoculation, 24 and 48 hpi), were assembled v

with paired-end reads and gene expression profiles compared in resistant and susceptible plants at each time point. Gene expression was notably different in resistant versus susceptible phenotypes. Resistant plants significantly up-regulated 185 genes 48 hours after pathogen exposure, while susceptible plants only up-regulated three genes. Most significantly up-regulated in resistant plants were gene homologues coding for transcription factors, receptor-like kinases and enzymes involved in the secondary metabolite pathway and in defense. Chapter Four: Resistant Syzygium luehmannii plants respond to Austropuccinia psidii challenge with up-regulation of putative chitinase and receptor genes Two manuscripts (Christie et al., 2016; Tobias et al., 2017) published during the PhD tenure formed the background to the research in this chapter. Manuscripts are attached as Appendices (Appendix 6 and 7), however publication paragraphs are utilized within the introduction and research findings are cited throughout this chapter. Though different patterns of gene expression followed inoculation of resistant or susceptible Syzygium luehmannii plants, the differential expression was observed by creating a single combined transcriptome, and then mapping reads from each plant and at each time point following inoculation. To test if individual plants harbor different transcript variants or different counts to transcripts, which may have been lost during the merging of transcriptomes, two important gene families involved in plant defence, the chitinases and nucleotide-binding site leucine-rich repeat receptors (NBS-LRR), were scrutinized further within each transcriptome. Species-specific nucleotide Hidden Markov Models were built and these were used to screen transcriptomes for potential variant genes and isoforms. The subsequent lists of transcripts were then translated and domains identified. All plants expressed multiple ‘genes’ and ‘isoforms’ of NBS-LRRs and the most prevalent additional domains included Toll or interleukin-1 (TIR), LRR vi

and Jacalin. The up-regulated predicted NBS-LRR transcript in resistant plants is identified as a TIR-NBS-LRR with homology to Eucalyptus grandis predicted genes on chromosome three. Chitinase ‘genes’ and ‘isoforms’ conformed with previously identified glycosyl hydrolase (GH) 18 and GH19 domains. Translated GH19 sequences, including the highly significantly upregulated Class II chitinase within resistant plants, indicate a high degree of conservation. Comparisons of counts, based on transcripts of interest, within the merged transcriptome versus individual transcriptomes show that gene expression trends are concordant. Chapter Five: Syzygium luehmannii transcripts within the defence functional pathway validated with PCR amplification and sequencing Homologues of genes known to be important in the defence functional pathway were significantly differentially expressed. These included gene homologues for receptor-like kinases, NBS-LRRs, transcription factors and defence-related proteins. To test for sequence variation within functional defence-related genes from resistant versus susceptible plants, primers were designed and putative genes were amplified. Amplicons of Class II chitinases, Class IA chitinase, thaumatin-like protein, disease resistance response protein, strigolactone receptor D14, respiratory burst oxidase, leucine rich repeat-extensin-like protein and Myb46 transcription factor show the presence of genes within each plant. Aligned amplicon sequences of two Class II chitinases, disease resistance response protein, strigolactone receptor D14 and Myb46 transcription factor show them to be homologous thereby indicating that downstream defence mechanisms are present in all plants. Primers for the differentially expressed putative NBS-LRR transcript were not successful, indicating a more comprehensive assembly of these recognition genes is required. These receptor-like genes are likely to be important in successful

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pathogen recognition and triggering downstream processes and therefore future studies should focus on characterisation and validation of these gene sequences. Conclusions arising from the research and future directions A concluding chapter provides key findings and future directions arising from the research.

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Table of Contents 1 A curious case of resistance to a new encounter pathogen: the Australian myrtle rust example ...................................................................................................................................... 1 1.1

Abstract ............................................................................................................................ 1

1.2

The plant pathogen co-evolution paradigm ...................................................................... 1

1.3

Myrtle rust in Australia .................................................................................................... 3

1.4

Host jump or long association? ........................................................................................ 7

1.5

Could ancient genes survive the ravages of time? ........................................................... 8

1.6

Receptor pairs and complexes ........................................................................................ 10

1.7

Guard or decoy strategies across a whole plant family? ................................................ 12

1.8

Concluding remarks ....................................................................................................... 14

1.9

Acknowledgements ........................................................................................................ 14

1.10

References ...................................................................................................................... 14

2 Screening two wild-sourced Australian myrtaceous species for resistance to Austropuccinia psidii (myrtle rust) .................................................................................................................... 21 2.1

Abstract .......................................................................................................................... 21

2.2

Introduction .................................................................................................................... 21

2.3

Methods .......................................................................................................................... 25

2.3.1

Chamelaucium uncinatum plant material ....................................................................... 25

2.3.2

Syzygium luehmannii plant material ............................................................................... 27

2.3.3

Inoculation with Austropuccinia psidii .......................................................................... 28

2.3.4

Scoring for infection....................................................................................................... 29

2.3.5

Challenge inoculation ..................................................................................................... 29

2.3.6

Scanning electron microscope images ........................................................................... 30

2.4

Results ............................................................................................................................ 31

2.4.1

Chamelaucium uncinatum infection responses .............................................................. 31

2.4.2

Syzygium luehmannii infection responses ...................................................................... 32

2.4.3

Syzygium luehmannii responses to challenge inoculation .............................................. 34

2.5

Discussion ...................................................................................................................... 35

2.6

Conclusion...................................................................................................................... 38

2.7

References ...................................................................................................................... 38

3 Identification of genes involved in resistance to Austropuccinia psidii (myrtle rust) in Syzygium luehmannii (Riberry) ................................................................................................ 41 3.1

Abstract .......................................................................................................................... 41 ix

3.2

Introduction .................................................................................................................... 41

3.3

Methods .......................................................................................................................... 43

3.3.1

Leaf samples collected and RNA extracted ................................................................... 43

3.3.2

RNA library preparation for sequencing ........................................................................ 44

3.3.3

Transcriptome assembly................................................................................................. 45

3.3.4

Alignment ....................................................................................................................... 46

3.3.5

Gene expression analysis ............................................................................................... 46

3.3.6

Differential expression data visualization ...................................................................... 47

3.4

Results ............................................................................................................................ 47

3.4.1

Transcriptome assembly................................................................................................. 47

3.4.2

Read counts mapped to merged Syzygium transcriptome ............................................. 49

3.4.3

Expression differences in resistant versus susceptible plants ........................................ 49

3.4.4

Expression changes in susceptible plants at 24 and 48 hours ........................................ 53

3.4.5

Expression changes in resistant plants at 24 and 48 hpi ................................................ 54

3.5

Discussion ...................................................................................................................... 57

3.5.1

Resistant plants recognize the pathogen......................................................................... 58

3.5.2

Resistant plants respond actively to the pathogen .......................................................... 59

3.5.3

Resistant plants produce secondary metabolites and cell wall fortification................... 61

3.5.4

Susceptible plants have no response to the pathogen ..................................................... 62

3.5.5

Explaining the results ..................................................................................................... 62

3.6

Conclusion...................................................................................................................... 64

3.7

References ...................................................................................................................... 64

4 Resistant Syzygium luehmannii plants respond to Austropuccinia psidii challenge with upregulation of putative chitinase and receptor transcripts ......................................................... 70 4.1

Abstract .......................................................................................................................... 70

4.2

Introduction .................................................................................................................... 71

4.3

Methods .......................................................................................................................... 74

4.3.1

Building species-specific Hidden Markov Models ........................................................ 74

4.3.2

Differential expression of NB-ARC domain containing transcripts visualized ............. 75

4.3.3

Gene sequences examined for protein domains ............................................................. 75

4.3.4

Chitinase classes identified ............................................................................................ 76

4.3.5

NBS-LRR and chitinase expression count data from individual transcriptomes ........... 76

4.3.6

Alignments and phylogenetic analysis ........................................................................... 77

4.3.7

NBS-LRR predicted protein structure ............................................................................ 78

4.4

Results ............................................................................................................................ 78 x

4.4.1

Predicted NBS-LRR genes and isoforms within Syzygium luehmannii ......................... 78

4.4.2

Up-regulated NBS-LRR in resistant plants .................................................................... 80

4.4.3

Predicted chitinase genes within Syzygium luehmannii ................................................. 85

4.4.4

Counts for up-regulated transcripts within individual plants ......................................... 90

4.5

Discussion ...................................................................................................................... 94

4.5.1

Novel fused NBS-LRR domains indicate potential for pathogen recognition ............... 94

4.5.2

A putative resistance gene candidate identified within Syzygium luehmannii ............... 95

4.5.3

Multiple isoforms of NBS-LRR within Syzygium luehmannii transcriptomes .............. 96

4.5.4

Chitinases are highly conserved within Syzygium luehmannii....................................... 97

4.5.5

Novel fused chitinase domains ....................................................................................... 98

4.5.6

Transcript counts based on the merged transcriptome shows accurate trends ............... 98

4.6

Conclusion...................................................................................................................... 99

4.7

References .................................................................................................................... 100

5 Syzygium luehmannii transcripts within the defence functional pathway validated with PCR amplification and sequencing ................................................................................................. 105 5.1

Abstract ........................................................................................................................ 105

5.2

Introduction .................................................................................................................. 105

5.3

Methods ........................................................................................................................ 106

5.3.1

Differentially expressed genes selected and primers designed .................................... 106

5.3.2

cDNA reverse transcribed from mRNA ....................................................................... 108

5.3.3

Testing the primers ....................................................................................................... 108

5.3.4

Sequencing the amplicons ............................................................................................ 109

5.4

Results .......................................................................................................................... 109

5.4.1

Primers successfully amplify products ......................................................................... 109

5.4.2

Amplicon sequences aligned ........................................................................................ 110

5.4.3

Amplicon match with transcriptomes .......................................................................... 117

5.5

Discussion .................................................................................................................... 119

5.6

Conclusion.................................................................................................................... 120

5.7

References .................................................................................................................... 120

6 Conclusions arising from the research and future directions ................................................. 122 6.1

Introduction .................................................................................................................. 122

6.2

The plant: pathogen interaction .................................................................................... 122

6.3

Recognition is the key to resistant phenotypes ............................................................ 123

6.4

Defence gene transcripts identified within Syzygium luehmannii ................................ 125

6.5

More data and a more comprehensive assembly.......................................................... 126 xi

6.6

Concluding statement ................................................................................................... 127

6.7

References .................................................................................................................... 128

Table of Tables Table 2.1 Chamelaucium uncinatum cuttings were collected from population locations north of Perth, WA ............................................................................................................................ 25 Table 2.2 Austropuccinia psidii urediniospore accessions used for screening plants. A presumed single pathotype in Australia is labelled 622 and G(-) for greenhouse (increase)... 29 Table 2.3 Scoring methodology for Austropuccinia psidii infection on Myrtaceae species (Morin et al., 2012) .................................................................................................................. 29 Table 2.4 Responses of Syzygium luehmannii plants to challenge inoculation with Austropuccinia psidii. .............................................................................................................. 34 Table 3.1. Basic raw statistics and from Trinity assembly of Illumina Hiseq2500 sequenced mRNA extracted from eight biological replicates of Syzygium luehamnnii. Resistant plants shaded. ...................................................................................................................................... 48 Table 3.2 Total read counts mapped to merged Syzygium assembly ........................................... 49 Table 4.1 NB-ARC-type ‘genes’ within individual Syzygium luehmannii plants, inoculated with Austropuccinia psidii, from which putative isoforms arise. R = resistant, S = susceptible, SL = combined transcriptome. ............................................................................. 80 Table 4.2 Isoform numbers of the up-regulated tobacco mosaic virus resistance gene N homologue (APR) within individual and merged. Syzygium luehmannii transcriptomes following inoculation with Austropuccinia psidii. R = resistant, S = susceptible, SL = merged transcriptome. .............................................................................................................. 82 Table 5.1 Primers designed and tested for amplification of Syzygium luehmannii cDNA following inoculation with Austropuccinia psidii. Shaded cells indicate no successful amplification product. ............................................................................................................ 110 Table 5.2 Amplicon sequences from Syzygium luehmannii plants with resistant (R1-3) and susceptible (S1-3) response to inoculation with Austropuccinia psidii were queried against the individual plant transcriptomes and against the Eucalyptus grandis genome (v2.0) for best matches using BLAST. Grey sequence IDs appear to be either incomplete or misassembled transcripts. ....................................................................................................... 118

Table of Figures Figure 1.1 Percent of A. psidii susceptible individuals (n=10) in controlled inoculation within taxa of four Australian Myrtaceae tribes; Tristanieae (white), Syzygieae (check), Leptospermeae (black), Eucalypteae (grey) (subset of data from Morin et al., 2012). Plants were considered susceptible with scores of 3-5 based on Morin et al. (2012). ......................... 6

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Figure 2.1 (A) Current locations for occurrence of Austropuccinia psidii within Australia (modified from Plant Biosecrutiy CRC Queensland Govt website 2016). (B) Relative climate suitability as indicated by the CLIMEX Ecoclimatic Index (map from Kriticos et al., 2013) with red indicating area optimal for A. psidii growth and survival year round and white no establishment potential. ............................................................................................. 22 Figure 2.2 Mapped natural distribution of Chamelaucium uncinatum (A) and Syzygium luehmannii (B) based on records from the Atlas of Living Australia (http://www.ala.org.au/). .......................................................................................................... 24 Figure 2.3 (A) Chamelaucium uncinatum (Geraldton waxflower) specimens were collected from locations north of Perth, WA as indicated by black diamonds, using co-ordinates in GoogleMaps. (B) Syzygium luehmanni (Riberry) were commercially propagated using fruit collected from parent trees located at Pottsville and Brunswick Heads, NSW as indicated by black diamonds.................................................................................................................... 27 Figure 2.4 Chamelaucium uncinatum plants showed no sign of resistance to controlled inoculation with Austropuccinia psidii. Pustules were present on all specimens 14 days post inoculation. Photographs were taken 15 days post inoculation ........................................ 32 Figure 2.5 Percent of resistant and susceptible Syzygium luehmannii plants in response to inoculation with Austropuccinia psidii based on the current study (103 plants, grey bars) and previous study (10 plants, black bars (Morin et al., 2012)) and responses of plants that were previously field infected (17 plants, light grey bars). Scores from 1 (highly resistant) – 5 (highly susceptible) based on system developed by Morin et al. (2012). .......................... 33 Figure 2.6 Responses of Syzygium luehmannii plants to controlled inoculation with Austropuccinia psidii. Resistance, indicated by localised necrotic regions, in samples A, B and C. Susceptible plants developed pustules, samples D, E, and F. Photographs were taken 21 days post inoculation. Scale bar ~ 10 mm. ................................................................ 33 Figure 2.7 Scanning electron microscopy (A-C) and light microscopy (D, E) of Austropuccinia psidii spore germination and penetration of leaf surfaces 48 hours following challenge inoculation of Syzygium luehmannii. A and E = previously resistant plants, B, C and D = previously susceptible plants. Ap = appressorium, U = urediniospore. Scale bar = 10 µm for SEM and 20 µm light microscopy images. .......................................... 35 Figure 3.1 Significant differential expression (FDR