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)

1. Lovejoth

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. Wheat is one of the first cereals known to have been domesticated, and wheat's ability
to self-pollinate greatly facilitated the selection of many distinct domesticated varieties.

4. “RUST NEVER DIES”

5. Two classes of pathogens based on interactions
with 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. Different Rust Hosts
Rusts are plant diseases caused by pathogenic fungi of the order Pucciniales. About
7800 species are known, Phylum: Basidiomycota Class: Pucciniomycetes

7. Wheat Rust
Leaf rust: Puccinia triticina
Stripe rust :Puccinia striiformis
f.sp. tritici
Stem rust:Puccinia graminis f.sp.
tritici

8. Urediospores are responsible for disease outbreak
MACROCYCLIC,MICROCYCLIC (always have an autoecious ) and DEMICYCLIC

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 italica
cycle,one is economic host
Autoecious: fungus which can complete its life cycle on a single host
species

10. Resistance is broadly categorized into two
groups
Race-specific type, gene-for-gene resistance= Controlled by genes with major
to intermediate effects, is short lived, often lasting for an average of about five
years when deployed. In wheat life of effective race-specific resistance genes
can be prolonged by using gene combinations. A majority of the genes follow
the gene-for-gene concept (Flor, 1956)
Race-nonspecific type (Polygenic, horizontal, general, minor, partial, slow
rusting and residual). When present alone, APR genes do not confer adequate
resistance 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. Immune Responses in Plants
In the coevolution of host-microbe interactions, pathogens acquired the ability to deliver effector proteins to the plant cell to
suppress PTI, allowing pathogen growth and disease. In response to the delivery of pathogen effector proteins, plants acquired
surveillance proteins (R proteins) to monitor the presence of the pathogen effector proteins
(Dodds et al.,2010)

12. Different Pathways Triggered on Pathogen Invasion
Additional modulation of the defense response is brought about by the effects
of a third signal transduction cascade triggered by ethylene (ET) produced upon
attack .Each of the signal-transduction pathways acts to activate a distinct set
of defense genes
(Koornneef et al., 2008)

13. Genes Triggered by Different Pathways
VEGETATIVE 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. Plant Response To Fungal Pathogen
Lincoln Taiz and Eduardo Zeiger

15. Cross Talk Between ETI and PTI
(Jun Liu et al.,2008)

16. COMPATIBLE INTERACTIONS(Susceptibility)
Susceptible wheat leaves (Mingxian 169) inoculated with Puccinia striiformis on the fifth day after
inoculation. Haustoria developed normally, mitochondria and nuclei normal ,no adverse effect on host
cell after haustoria formation ,haustorial protoplasm was well preserved and organelles were regularly
arranged, EHM continuous and undulated.
(Ma et al.,2009)

17. INCOMPATIBLE INTERACTIONS(Resistance)
Intercellular hypha with vacuolated mitochondria ,hyphal wall thickening and deeply
stained ,haustorial mother cell vacuolated with deeply stained lipid material ,haustorium
surrounded by callose and necrotized, extrahaustorial membrane wrinkled, extrahaustorial
matrix thickened with electron-dense material deposited, host organelles disintegrated.
(Ma et al.,2009)

18. Cytological differences in compatible and incompatible
interactions
Susceptibility Resistance
(Ma et al.,2009)

19. Most genes confer race-specific resistance in a
gene-for-gene manner. 1900 - 1955 various major
resistance genes were discovered.
Erosion of race specific resistance genes, or their
combinations
 Wheat varieties relying on race-specific
resistance lose effectiveness
within a few years.
 Adult plant resistance is believed to be more
durable but it is more difficult to evaluate, the
multigenic nature of APR impedes the use of MAS
efforts
 Adaptability of fungi due to enormous genetic
fluidity, result in genomic rearrangements and
mutations, migration and adaptation of the fungus
to the diverse climatic conditions where wheat is
grown
(Bakkeren et al.,2012)

20. Need of studying HPI?
 To reveal Signal transduction events and expression of
disease resistance.
 Understand molecular basis and evolution of R gene
specificity.
 Evolution of plant disease resistance to specific
pathogen.
 Cloning and characterization of plant R genes.
 Identification of novel and stable plant resistance genes
How functional genomics can help?
Identification of fungal virulence genes or host response
elements, which can be useful for breeding programmes.
Large-scale genomic projects will reveal many PAMPs
(Bakkeren et al.,2012)

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 (including
RFLPs, SSRs, AFLPs, SNPs, and DArT markers), construction of molecular genetic and
physical 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 been
utilized successfully, and a number of genes/QTL have been cloned. The first genes to be
isolated from wheat by map-based cloning included three resistance genes, including
leaf rust Lr21; and Lr10; and powdery mildew Pm3b
 Organellar genomes including chloroplast and mitochondrial genomes have been fully
sequenced
(Gupta.,2009)

22. Puccinia graminis f. sp. tritici genomic
89-Mb genome ofby Sanger whole-genome
shotgun strategy.
No evidence for whole-genome duplication or
large-scale dispersed segmental duplications ,the
expanded size results from a massive proliferation of
transposable elements.
Predicted 17,773 protein coding genes.
Genomic features related to their obligate
biotrophic lifestyle include expanded lineage-specific
gene families, a large repertoire of effector-like small
secreted proteins, impaired nitrogen and sulfur
assimilation pathways, and expanded families of
amino acid and oligopeptide membrane transporters
.
(Sebastein et al.,2011)

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. Gene expression profiling can be divided into three
categories:
1 PCR: RT-PCR (qualitative and quantitative),DDRT-PCR
2) Sequencing based :cDNA sequencing (full-length
cDNAs, subtracted cDNAs, normalized cDNA
libraries), SAGE, Massive parallel signature sequencing
,454 and Solexa.
3) Hybridization based: Northern
blots, Macroarrays, DNA microarrays, Oligonucleotide
microarrays, Differential display, cDNA-AFLP
Deep sequencing and whole genome tilling
arrays are becoming increasing important
(Yunbi Xu.,2010)

25. Interaction transcriptomics reveals key plant and microbial genes that play important roles during
these 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. cDNA Sequencing
Results in high-copy-number mRNAs being overrepresented whereas low-copy-number mRNAs are missed. NGS do
not require cDNA molecules to be cloned before sequencing ,however, they provide much shorter reads than are
obtained by Sanger sequencing. Ultra-high-throughput sequencing will yield reads from more mRNAs, complete
transcripts will have to be assembled from many short reads.

27. For cereal rust fungi, microarrays (Duplessis et al., 2011) and EST/cDNA arrays
have yielded information on genes expressed during infection. The use of the
Wheat GeneChip® technique is often conditioned by known gene sequences.
with limited ESTs unspecific to different wheat materials

28. Transcriptome analysis of the wheat–Puccinia striiformis f. sp. tritici interaction
To identify transcripts associated with the Yr5 –mediated incompatible interaction and the
Yr5 -compatible interaction, the Wheat GeneChip was used to profile the changes occurring in
wheat isolines gene after inoculation with Pst. Gene Chip represents over 55 000 wheat transcripts
from all chromosomes and ancestral genomes. The temporal pattern of transcript accumulation
showed 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 response
involving signalling pathways and defence-related transcripts ,protein kinase signalling , reactive
oxygen species. To facilitate the map-based cloning of Yr5 , the GeneChip data was explored for the
development of genetic markers that were linked to Yr5
(Coram et al.,2008)

29. Differential Display rtPCR
Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis
of gene expression ,identification of virulence factors, genes involved in cell death, and signaling genes
(Peng Liang.,2002)

30. Genes associated with resistance to wheat yellow rust disease identified by
differential display analysis
Differential display reverse transcriptase-PCR method (DDRT-PCR) was used on two of the
yellow rust differential lines of wheat, infected with the virulent and the avirulent Puccinia
striiformis 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. Genes detected may have roles in ubiquitinylation, programmed cell death , putative antifungal
activities, disease resistance responses, pathogenesis related responses, plant disease resistance like
genes, pathogen related genes, and a gene with putative antifungal activity. Genes involved in ubiquitin
mediated protein degradation are regulated in wheat in response to yellow rust incompatible pathogen
infection and suggest that ubiquitinylation and protein degradation are significant regulatory mechanisms in
wheat yellow rust disease resistance
(Osman et al.,2008)

32. Resistance mechanism may be diverging in these two plants
(Osman et al.,2008)

33. (SSH) is a PCR-based cDNA subtraction method, and it
selectively amplify target cDNA fragments (differentially
expressed) and simultaneously suppress nontarget DNA
amplification.
( Gadgil et al .,2002)

34. Gene Expression in Wheat Induced by Inoculation with Puccinia striiformis West
A (SSH) cDNA library was constructed from Pst infected seedling leaves of an immune resistant
germplasm Shaanmai 139. A total of 84 ESTs were obtained. BlastX searches identified 45 of the
Unigenes 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 genes
that are induced by cold. The genes of known function include those with potential biological
roles in signal transmission, energy and metabolism, transcription regulation, phenyl propanoid
pathway, and defense response.
(Hong et al .,2011)

35. Senescence-associated genes, omega-6 fatty
acid desaturase, and acyl-CoA synthetase
showed bimodial pattern suggesting complex
patterns of defense related gene expression in
multi resistance gene cultivars.
(Hong et al .,2011)

36. LongSAGE, RL-SAGE and SuperSAGE
The basis of the technique is that these 12 bp sequences, despite their shortness, are sufficient to enable the gene that
codes for the mRNA to be identified. SAGE sampling is based on sequencing mRNA output, not on hybridization of
mRNA output to probes, so transcription levels are measured more quantitatively than by microarray.

37. Magnaporthe grisea (blast)-infected rice leaves, gene
expression profiles of both the rice host and blast fungus
was simultaneously monitored ,revealing that the
hydrophobin gene is the most actively transcribed M.
grisea gene in blast-infected rice leaves.
SuperSAGE has been applied to study gene expression
changes elicitor-treated Nicotiana benthamiana, a
‘‘nonmodel’’ organism for which no DNA
database is available. SuperSAGE allowed rapid
identification of genes up- or down-regulated by the
elicitor.
(Matsumura.,2003)

38. (Matsumura et al.,2003)

39. cDNA AFLP does not require prior sequence information and is universal for any organisms or
interactions, and is, therefore, a powerful tool for identifying novel genes in non-model
organisms like wheat

40. Differential gene expression in incompatible interaction between wheat and
stripe rust fungus revealed by cDNA-AFLP and comparison to compatible
interaction
52,992 transcript derived fragments (TDFs) were generated with 64 primer pairs and
2,437 of them displayed altered expression patterns after inoculation , 1,787 up-regulated
and 650 down-regulated. 161 TDFs were shared by both interactions, 94 were expressed
specifically in the incompatible interaction .A large group (17.6%) of these genes shared
high homology with genes involved in metabolism and photosynthesis; 13.8% to genes
with functions related to disease defense and signal transduction; and those in the
remaining groups (12.9%) to genes involved in transcription, transport processes, protein
metabolism, and cell structure.
(Wang et al.,2010)

41.  Roche/454 FLX: 2004
 Illumina Solexa Genome Analyzer: 2006
 Applied Biosystems SOLiDTM System: 2007

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. The DNA sample of interest is sheared to
appropriate size (average ~800bp) using
a compressed air device known as a
nebulizer. The flow cell surface is coated
with single stranded oligonucleotides
that correspond to the sequences of the
adapters, repeated denaturation and
extension results in localized
amplification of single molecules in
millions of unique locations across the
flow cell surface. This process occurs
"cluster station“.

44. A flow cell containing
millions of unique clusters
is now loaded into the 1G
sequencer for automated
cycles of extension and
imaging.

45. Differences in Platforms

46. Gene Expression in Leaves of Susceptible Glycine max during Infection with
Phakopsora pachyrhizi Using Next Generation Sequencing
cDNA libraries were constructed from RNA isolated from whole infected soybean leaves 10 days after inoculation
with P. pachyrhizi and sequenced using an Illumina platform ,15 million sequences corresponding to soybean
genes were obtained. 42% of the genes were down regulated including genes encoding proteins involved in
amino acid metabolism, carbohydrate metabolism, and transport facilitation; 31% were upregulated including
genes 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. (Tremblay.,2011)

48. (Tremblay.,2011)

49. Why Proteomics to Study Host
Pathogen Interactions
Global study of the protein content of a cell
mRNA abundance are not always
mirrored by corresponding protein
levels .An mRNA produced in
abundance may be degraded
rapidly or translated inefficiently
Many transcripts give rise to
more than one proteins

50. Proteomics Work Flow
Isotope Coded Affinity Tags (ICAT) the isotopes are in affinity tags, Stable isotope labeling by amino acids
in cell culture (SILAC) uses stable isotopes to label amino acids , Isotope tagged relative and absolute
quantitation (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 many
predicted pathogenicity and virulence factors.
(B.F. Quirino et al.,2010)

51. Protein phosphorylation is a pivotal process during
plant–pathogen interactions
Proteins undergo phosphorylation /cleaved or degraded
(B.F. Quirino et al.,2010)

52. Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible
host-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. Peptides
from 32 proteins were analyzed.
(Rampitsch et al. ,2006)

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. 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 proteasome
Metabolic enzymes (e.g., carbohydrate Involved in protecting the fungus from general
kinase); structural proteins (alpha-tubulin stress generated by host defense
and ribosomal protein); heat shock mechanisms, combating a plant-induced oxidative
proteins, ascorbate peroxidase burst
(Rampitsch et al. ,2006)

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. METABOLOMICS
Metabolomics comprehensive identification and quantification of all low
molecular weight metabolites and their relationships in a biological sample at
a specific time point.
Metabolomics provides information about the ultimate biochemical outcome
of changes in the genome, transcriptome and proteome.

57. Steps and Techniques Involved In Metabolomics
Mass spectrometry utilizes the retention time for a given metabolite, as well as the mass/charge ratio (MS), often in conjunction with
the mass/charge ratio of fragmentation daughter ions in tandem MS (MS/MS), for identification. NMR exploits structurally dependent
changes in the magnetic resonance of suitable nuclei for metabolite identification. Light spectroscopic approaches do not perform as
well for the individual identification of metabolites but represent a more cost-effective approach to determining changes in the overall
metabolic profile or fingerprint of a sample.
(Kafsack et al.,2010)

58. Investigation of interactions in the pathosystem potato (Solanum tuberosum L.) and
Rhizoctonia 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. Potato sprout metabolome ,selected sub-networks of amino acids and glycoalkaloids .Nodes represent
metabolites, enzymes or chemical reactions. Changes in more than 300 identified potato sprout metabolites in
response to pathogen attack was detected .Discovery of biomarkers that could be exploited in plant
breeding, and applications in biotechnology and/or crop protection
(Konstantinos A. Aliferis & Suha Jabaji.,2012)

60. Challenges in metabolome studies during plant pathogen interaction
Presence of fungal-derived metabolites
Lack of unifying principles such as genetic code
Solutions
Use of fungal metabolite profiles.
 The construction of comprehensive metabolite databases for fungal
metabolites.
(Konstantinos A. Aliferis a & Suha Jabaji.,2012)

61. Understanding the developmental and physiological adaptations of pathogens
that allow them to invade plants, colonize tissues, and subvert plant metabolism is
considerable challenge.
It is important to develop an understanding of the molecular basis of pathogen
recognition by plants, which underlies the evolution of disease resistance.
Combining information derived from metabolomics, proteomics and
transcriptomics will help to understanding of the wheat rust pathosystem.