Functional Genomics of Plant Pathogen interactions in Wheat Rust Pathosystem

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Cereal rust fungi are pathogens of major importance to agriculture, threatening cereal production worldwide. Targeted breeding for resistance, based on information from fungal surveys and population structure analyses of virulence, has been effective. Nevertheless, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to determine the range of available resistance genes. The development of genomic resources for these fungi and their comparison has released a torrent of new ideas and approaches to use this information to assist pathologists and agriculture in general. The sequencing of gene transcripts and the analysis of proteins from haustoria has yielded candidate virulence factors among which could be defence-triggering avirulence genes. Genome-wide computational analyses, including genetic mapping and transcript analyses by RNA sequencing of many fungal isolates, will predict many more candidates (Bakkeren et al., 2012)

Dissecting the mechanisms of host-pathogen systems like wheat-rust, including pathogen counter-defenses will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi (Hadrami et al.,2012)

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Functional Genomics of Plant Pathogen interactions in Wheat Rust Pathosystem

  1. 1. Lovejoth
  2. 2. Rust and their life cycleDefense employed by host plant Use of functional genomics in studying host pathogen interactionsTranscriptomics as a tool to study host pathogen interactionsProteomics as a tool to study host pathogen interactionsMetabolomic approaches for studying host pathogen interactions
  3. 3. Wheat is one of the first cereals known to have been domesticated, and wheats abilityto self-pollinate greatly facilitated the selection of many distinct domesticated varieties.
  4. 4. “RUST NEVER DIES”
  5. 5. Two classes of pathogens based on interactionswith plants Biotrophic fungal pathogens colonize living plant tissue and obtain nutrients from living host cells, invade only a few of the cells, it can reduce the competitive abilities of the host. Hemibiotrophs derive nutrients from a combination of feeding from living and killed host cells Necrotrophic fungal pathogens infect and kill host tissue and extract nutrients from the dead host cells. “Biotrophic parasitism is evolutionarily advanced” Rice Blast Powdery Mildew
  6. 6. Different Rust HostsRusts are plant diseases caused by pathogenic fungi of the order Pucciniales. About7800 species are known, Phylum: Basidiomycota Class: Pucciniomycetes
  7. 7. Wheat Rust Leaf rust: Puccinia triticina Stripe rust :Puccinia striiformis f.sp. triticiStem rust:Puccinia graminis f.sp.tritici
  8. 8. Urediospores are responsible for disease outbreakMACROCYCLIC,MICROCYCLIC (always have an autoecious ) and DEMICYCLIC
  9. 9. . Meadow Rue Barberry Alternate host of stem rust of Triticum turgidium Alternate host of leaf rust of Triticum turgidium and Triticum aestivum and Triticum aestivum Heteroecious :require two unrelated hosts to complete their life Anchusa italicacycle,one is economic host Autoecious: fungus which can complete its life cycle on a single hostspecies
  10. 10. Resistance is broadly categorized into twogroupsRace-specific type, gene-for-gene resistance= Controlled by genes with majorto intermediate effects, is short lived, often lasting for an average of about fiveyears when deployed. In wheat life of effective race-specific resistance genescan be prolonged by using gene combinations. A majority of the genes followthe gene-for-gene concept (Flor, 1956)Race-nonspecific type (Polygenic, horizontal, general, minor, partial, slowrusting and residual). When present alone, APR genes do not confer adequateresistance especially under high disease pressure; however, combinations of 4–5 such genes usually result in ‘‘near-immunity’’ or a high level of resistance. (Singh et al.,2010)
  11. 11. Immune Responses in PlantsIn the coevolution of host-microbe interactions, pathogens acquired the ability to deliver effector proteins to the plant cell tosuppress PTI, allowing pathogen growth and disease. In response to the delivery of pathogen effector proteins, plants acquiredsurveillance proteins (R proteins) to monitor the presence of the pathogen effector proteins (Dodds et al.,2010)
  12. 12. Different Pathways Triggered on Pathogen InvasionAdditional modulation of the defense response is brought about by the effectsof a third signal transduction cascade triggered by ethylene (ET) produced uponattack .Each of the signal-transduction pathways acts to activate a distinct setof defense genes (Koornneef et al., 2008)
  13. 13. Genes Triggered by Different PathwaysVEGETATIVE STORAGE PROTEIN 1 (VSP1) by JA, GLUTAREDOXIN 480 (GRX480) by SA,and PATHOGENESIS-RELATED 3/CHITINASE B (PR-3/ChiB) and PR-4/HEVEIN-LIKE (HEL)by ET. PLANT DEFENSIN 1.2 (PDF1.2) by ET and JA, or PR-1 and GLUTATHION-S-TRANSFERASE 1 (GST1) by ET and SA. (Verk et al.,2010)
  14. 14. Plant Response To Fungal Pathogen Lincoln Taiz and Eduardo Zeiger
  15. 15. Cross Talk Between ETI and PTI (Jun Liu et al.,2008)
  16. 16. COMPATIBLE INTERACTIONS(Susceptibility)Susceptible wheat leaves (Mingxian 169) inoculated with Puccinia striiformis on the fifth day afterinoculation. Haustoria developed normally, mitochondria and nuclei normal ,no adverse effect on hostcell after haustoria formation ,haustorial protoplasm was well preserved and organelles were regularlyarranged, EHM continuous and undulated. (Ma et al.,2009)
  17. 17. INCOMPATIBLE INTERACTIONS(Resistance) Intercellular hypha with vacuolated mitochondria ,hyphal wall thickening and deeplystained ,haustorial mother cell vacuolated with deeply stained lipid material ,haustoriumsurrounded by callose and necrotized, extrahaustorial membrane wrinkled, extrahaustorialmatrix thickened with electron-dense material deposited, host organelles disintegrated. (Ma et al.,2009)
  18. 18. Cytological differences in compatible and incompatibleinteractions Susceptibility Resistance (Ma et al.,2009)
  19. 19. Most genes confer race-specific resistance in agene-for-gene manner. 1900 - 1955 various majorresistance genes were discovered.Erosion of race specific resistance genes, or theircombinations Wheat varieties relying on race-specificresistance lose effectivenesswithin a few years. Adult plant resistance is believed to be moredurable but it is more difficult to evaluate, themultigenic nature of APR impedes the use of MASefforts Adaptability of fungi due to enormous geneticfluidity, result in genomic rearrangements andmutations, migration and adaptation of the fungusto the diverse climatic conditions where wheat isgrown (Bakkeren et al.,2012)
  20. 20. Need of studying HPI? To reveal Signal transduction events and expression ofdisease resistance. Understand molecular basis and evolution of R genespecificity. Evolution of plant disease resistance to specificpathogen. Cloning and characterization of plant R genes. Identification of novel and stable plant resistance genesHow functional genomics can help?Identification of fungal virulence genes or host responseelements, which can be useful for breeding programmes.Large-scale genomic projects will reveal many PAMPs (Bakkeren et al.,2012)
  21. 21. Wheat GenomicsWheat (Triticum aestivum L.) with a large genome (16000 Mb) High proportion (∼80%) of repetitive sequences.Development of thousands of molecular markers (includingRFLPs, SSRs, AFLPs, SNPs, and DArT markers), construction of molecular genetic andphysical maps Development of more than 1 million ESTsDevelopment of BAC/BIBAC resources for individual chromosomes Functional genomics approaches like TILLING, RNAi, and epigenetics have beenutilized successfully, and a number of genes/QTL have been cloned. The first genes to beisolated from wheat by map-based cloning included three resistance genes, includingleaf rust Lr21; and Lr10; and powdery mildew Pm3b Organellar genomes including chloroplast and mitochondrial genomes have been fullysequenced (Gupta.,2009)
  22. 22. Puccinia graminis f. sp. tritici genomic89-Mb genome ofby Sanger whole-genomeshotgun strategy.No evidence for whole-genome duplication orlarge-scale dispersed segmental duplications ,theexpanded size results from a massive proliferation oftransposable elements.Predicted 17,773 protein coding genes.Genomic features related to their obligatebiotrophic lifestyle include expanded lineage-specificgene families, a large repertoire of effector-like smallsecreted proteins, impaired nitrogen and sulfurassimilation pathways, and expanded families ofamino acid and oligopeptide membrane transporters. (Sebastein et al.,2011)
  23. 23.  Development and application of global (genome-wide or system-wide) experimental approaches to assess gene function by making use of the information and reagents provided by structural genomics. (Hieter and Boguski 1997) Functional genomics includes a systematic analysis of mRNA and protein expression, exploration of gene product interactions and their influence on different phenotypical traits to define gene functions.
  24. 24. Gene expression profiling can be divided into threecategories:1 PCR: RT-PCR (qualitative and quantitative),DDRT-PCR2) Sequencing based :cDNA sequencing (full-lengthcDNAs, subtracted cDNAs, normalized cDNAlibraries), SAGE, Massive parallel signature sequencing,454 and Solexa.3) Hybridization based: Northernblots, Macroarrays, DNA microarrays, Oligonucleotidemicroarrays, Differential display, cDNA-AFLPDeep sequencing and whole genome tillingarrays are becoming increasing important (Yunbi Xu.,2010)
  25. 25. Interaction transcriptomics reveals key plant and microbial genes that play important roles duringthese and other as yet unknown interactions. Challenges include How to discriminate pathogen from host ESTs. Similarity searches to genome/cDNA sequences. (Peer M. Schenk et al.,2012)
  26. 26. cDNA SequencingResults in high-copy-number mRNAs being overrepresented whereas low-copy-number mRNAs are missed. NGS donot require cDNA molecules to be cloned before sequencing ,however, they provide much shorter reads than areobtained by Sanger sequencing. Ultra-high-throughput sequencing will yield reads from more mRNAs, completetranscripts will have to be assembled from many short reads.
  27. 27. For cereal rust fungi, microarrays (Duplessis et al., 2011) and EST/cDNA arrayshave yielded information on genes expressed during infection. The use of theWheat GeneChip® technique is often conditioned by known gene sequences.with limited ESTs unspecific to different wheat materials
  28. 28. Transcriptome analysis of the wheat–Puccinia striiformis f. sp. tritici interactionTo identify transcripts associated with the Yr5 –mediated incompatible interaction and theYr5 -compatible interaction, the Wheat GeneChip was used to profile the changes occurring inwheat isolines gene after inoculation with Pst. Gene Chip represents over 55 000 wheat transcriptsfrom all chromosomes and ancestral genomes. The temporal pattern of transcript accumulationshowed a peak at 24 h after infection that may reflect haustorial penetration by Pst at 16 h..Annotation revealed that the presence of Yr5 resulted in a rapid and amplified resistance responseinvolving signalling pathways and defence-related transcripts ,protein kinase signalling , reactiveoxygen species. To facilitate the map-based cloning of Yr5 , the GeneChip data was explored for thedevelopment of genetic markers that were linked to Yr5 (Coram et al.,2008)
  29. 29. Differential Display rtPCRDifferential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysisof gene expression ,identification of virulence factors, genes involved in cell death, and signaling genes (Peng Liang.,2002)
  30. 30. Genes associated with resistance to wheat yellow rust disease identified by differential display analysisDifferential display reverse transcriptase-PCR method (DDRT-PCR) was used on two of theyellow rust differential lines of wheat, infected with the virulent and the avirulent Pucciniastriiformis f. sp. tritici races together with appropriate control inoculations. Total of 90 primer combinations were used in DDRT-PCR reactions were generated with different time points ,60 differentially expressed bands were identified and excised from sequencing gels. Among them, 50 could be reamplified and 39 of these 50 were randomly selected to be cloned and sequenced. (Osman et al.,2008)
  31. 31. Genes detected may have roles in ubiquitinylation, programmed cell death , putative antifungalactivities, disease resistance responses, pathogenesis related responses, plant disease resistance likegenes, pathogen related genes, and a gene with putative antifungal activity. Genes involved in ubiquitinmediated protein degradation are regulated in wheat in response to yellow rust incompatible pathogeninfection and suggest that ubiquitinylation and protein degradation are significant regulatory mechanisms inwheat yellow rust disease resistance (Osman et al.,2008)
  32. 32. Resistance mechanism may be diverging in these two plants (Osman et al.,2008)
  33. 33. (SSH) is a PCR-based cDNA subtraction method, and itselectively amplify target cDNA fragments (differentiallyexpressed) and simultaneously suppress nontarget DNAamplification. ( Gadgil et al .,2002)
  34. 34. Gene Expression in Wheat Induced by Inoculation with Puccinia striiformis WestA (SSH) cDNA library was constructed from Pst infected seedling leaves of an immune resistantgermplasm Shaanmai 139. A total of 84 ESTs were obtained. BlastX searches identified 45 of theUnigenes as similar to those encoding proteins of known function and eight of unknown function.Blast EST analysis of these Pst-induced genes showed that they were mostly homologous to genesthat are induced by cold. The genes of known function include those with potential biologicalroles in signal transmission, energy and metabolism, transcription regulation, phenyl propanoidpathway, and defense response. (Hong et al .,2011)
  35. 35. Senescence-associated genes, omega-6 fattyacid desaturase, and acyl-CoA synthetaseshowed bimodial pattern suggesting complexpatterns of defense related gene expression inmulti resistance gene cultivars. (Hong et al .,2011)
  36. 36. LongSAGE, RL-SAGE and SuperSAGEThe basis of the technique is that these 12 bp sequences, despite their shortness, are sufficient to enable the gene thatcodes for the mRNA to be identified. SAGE sampling is based on sequencing mRNA output, not on hybridization ofmRNA output to probes, so transcription levels are measured more quantitatively than by microarray.
  37. 37. Magnaporthe grisea (blast)-infected rice leaves, geneexpression profiles of both the rice host and blast funguswas simultaneously monitored ,revealing that thehydrophobin gene is the most actively transcribed M.grisea gene in blast-infected rice leaves.SuperSAGE has been applied to study gene expressionchanges elicitor-treated Nicotiana benthamiana, a‘‘nonmodel’’ organism for which no DNAdatabase is available. SuperSAGE allowed rapididentification of genes up- or down-regulated by theelicitor. (Matsumura.,2003)
  38. 38. (Matsumura et al.,2003)
  39. 39. cDNA AFLP does not require prior sequence information and is universal for any organisms orinteractions, and is, therefore, a powerful tool for identifying novel genes in non-modelorganisms like wheat
  40. 40. Differential gene expression in incompatible interaction between wheat and stripe rust fungus revealed by cDNA-AFLP and comparison to compatible interaction52,992 transcript derived fragments (TDFs) were generated with 64 primer pairs and2,437 of them displayed altered expression patterns after inoculation , 1,787 up-regulatedand 650 down-regulated. 161 TDFs were shared by both interactions, 94 were expressedspecifically in the incompatible interaction .A large group (17.6%) of these genes sharedhigh homology with genes involved in metabolism and photosynthesis; 13.8% to geneswith functions related to disease defense and signal transduction; and those in theremaining groups (12.9%) to genes involved in transcription, transport processes, proteinmetabolism, and cell structure. (Wang et al.,2010)
  41. 41.  Roche/454 FLX: 2004 Illumina Solexa Genome Analyzer: 2006 Applied Biosystems SOLiDTM System: 2007
  42. 42. Illumina Genome Analyzer Originally developed by Solexa, now subsidiary of Illumina. Commercially available in 2006 It produces 8-12 million reads per sample of 36 bp length = 10 GB/week. Run takes 3 days for 7 samples. Low error rate, mostly base changes, few indels
  43. 43. The DNA sample of interest is sheared toappropriate size (average ~800bp) usinga compressed air device known as anebulizer. The flow cell surface is coatedwith single stranded oligonucleotidesthat correspond to the sequences of theadapters, repeated denaturation andextension results in localizedamplification of single molecules inmillions of unique locations across theflow cell surface. This process occurs"cluster station“.
  44. 44. A flow cell containingmillions of unique clustersis now loaded into the 1Gsequencer for automatedcycles of extension andimaging.
  45. 45. Differences in Platforms
  46. 46. Gene Expression in Leaves of Susceptible Glycine max during Infection with Phakopsora pachyrhizi Using Next Generation SequencingcDNA libraries were constructed from RNA isolated from whole infected soybean leaves 10 days after inoculationwith P. pachyrhizi and sequenced using an Illumina platform ,15 million sequences corresponding to soybeangenes were obtained. 42% of the genes were down regulated including genes encoding proteins involved inamino acid metabolism, carbohydrate metabolism, and transport facilitation; 31% were upregulated includinggenes encoding proteins involved in lipid metabolism and signal transduction. A mRNA-Seq strategy using the Illumina platform in susceptible Glycine max during Infection with soyabean rust. (Tremblay.,2011)
  47. 47. (Tremblay.,2011)
  48. 48. (Tremblay.,2011)
  49. 49. Why Proteomics to Study Host Pathogen Interactions Global study of the protein content of a cellmRNA abundance are not alwaysmirrored by corresponding proteinlevels .An mRNA produced inabundance may be degradedrapidly or translated inefficientlyMany transcripts give rise tomore than one proteins
  50. 50. Proteomics Work FlowIsotope Coded Affinity Tags (ICAT) the isotopes are in affinity tags, Stable isotope labeling by amino acidsin cell culture (SILAC) uses stable isotopes to label amino acids , Isotope tagged relative and absolutequantitation (iTRAQ) peptides derived from each sample are derivatized with amine-specific isobaric tags.Partial proteome of cereal rust fungi has been generated a, focusing on isolated haustoria from Pt-infected wheat (Song et al., 2011).Over 260 proteins were identified .Among the proteins are manypredicted pathogenicity and virulence factors. (B.F. Quirino et al.,2010)
  51. 51. Protein phosphorylation is a pivotal process duringplant–pathogen interactions Proteins undergo phosphorylation /cleaved or degraded (B.F. Quirino et al.,2010)
  52. 52. Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptiblehost-pathogen interaction Susceptible line of wheat was infected with a virulent race of leaf rust and compared to mock- inoculated wheat using 2-DE (with IEF pH 4–8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-TOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.Distribution of proteins excised from gels and analyzed by MS/MS, by putative origin. Peptidesfrom 32 proteins were analyzed. (Rampitsch et al. ,2006)
  53. 53. Wheat leaves inoculated with a virulent race of leaf rust (9 DPI)Wheat leaves mock inoculated with oil (9DPI). (Rampitsch et al., 2006)
  54. 54. Wheat Proteins and Fungal Proteins eIF5a and EF1b :in a rice-virus interaction Involved in the control of protein turnover EIF1b :in maize as a result of fungal attack Degrades unwanted proteins Alpha 4 subunit of the 20S proteasomeMetabolic enzymes (e.g., carbohydrate Involved in protecting the fungus from generalkinase); structural proteins (alpha-tubulin stress generated by host defenseand ribosomal protein); heat shock mechanisms, combating a plant-induced oxidativeproteins, ascorbate peroxidase burst (Rampitsch et al. ,2006)
  55. 55. Pathosystems genomes of both interaction partners have been fully sequenced:M. oryzae/rice, R. solanacearum/Arabidopsis, X. oryzae pv. oryzae/rice, P.syringae pv. oryzae/rice, X. campestris pv. campestris/Arabidopsis, P. syringae pv.tomato/ Arabidopsis and X. fastidiosa/grapevine.Proteomics studies with these pathosystemsprotein identificationassignment of a particular protein to plant or pathogen origin.understanding of different aspects of plant–pathogen interactions (B.F. Quirino et al.,2010)
  56. 56. METABOLOMICSMetabolomics comprehensive identification and quantification of all lowmolecular weight metabolites and their relationships in a biological sample ata specific time point.Metabolomics provides information about the ultimate biochemical outcomeof changes in the genome, transcriptome and proteome.
  57. 57. Steps and Techniques Involved In MetabolomicsMass spectrometry utilizes the retention time for a given metabolite, as well as the mass/charge ratio (MS), often in conjunction withthe mass/charge ratio of fragmentation daughter ions in tandem MS (MS/MS), for identification. NMR exploits structurally dependentchanges in the magnetic resonance of suitable nuclei for metabolite identification. Light spectroscopic approaches do not perform aswell for the individual identification of metabolites but represent a more cost-effective approach to determining changes in the overallmetabolic profile or fingerprint of a sample. (Kafsack et al.,2010)
  58. 58. Investigation of interactions in the pathosystem potato (Solanum tuberosum L.) andRhizoctonia solani using metabolomics The level of metabolites is determined by the properties and concentration of enzymes. Thus, the level of metabolites represents the molecular phenotype of a cell or organism in response to the genetic or environmental factors. Detected compounds includes a large number of primary and secondary metabolites belonging to amino, fatty and carboxylic acids, carbohydrates, and terpenoid and steroidal glycoalkaloids (Konstantinos A. Aliferis & Suha Jabaji.,2012)
  59. 59. Potato sprout metabolome ,selected sub-networks of amino acids and glycoalkaloids .Nodes representmetabolites, enzymes or chemical reactions. Changes in more than 300 identified potato sprout metabolites inresponse to pathogen attack was detected .Discovery of biomarkers that could be exploited in plantbreeding, and applications in biotechnology and/or crop protection (Konstantinos A. Aliferis & Suha Jabaji.,2012)
  60. 60. Challenges in metabolome studies during plant pathogen interactionPresence of fungal-derived metabolitesLack of unifying principles such as genetic codeSolutionsUse of fungal metabolite profiles. The construction of comprehensive metabolite databases for fungalmetabolites. (Konstantinos A. Aliferis a & Suha Jabaji.,2012)
  61. 61. Understanding the developmental and physiological adaptations of pathogensthat allow them to invade plants, colonize tissues, and subvert plant metabolism isconsiderable challenge.It is important to develop an understanding of the molecular basis of pathogenrecognition by plants, which underlies the evolution of disease resistance.Combining information derived from metabolomics, proteomics andtranscriptomics will help to understanding of the wheat rust pathosystem.

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