Pathogenomics

Dr.Y.S.ParmarUniversityofHorticulture&Forestry
Ankita
Department of Plant Pathology
H-2016-70-M
PL.PATH.591. MASTER’S SEMINAR (1+0)

Pathogenomics in Plant pathology

Outline of seminar
 Introduction
 History
 Techniques used
 Effectors
 Conclusion
 Pathogenomics with reference
to plant pathology

Terms that are frequently used
Genomic - It is the field of molecular biology, that deals with the study of
genomes, the complete set of genetic material within an organism
Marker gene-A genetic marker is a gene or DNA sequence with a known location
on a chromosome that can be used to identify individuals or species.
Effectors - Effectors are proteins expressed by plant pathogens to aid infection of
specific plant species. These molecules can alter plant processes and are central to
understanding the complicated interplay between plants and their pathogens
Probe – It is a fragment of DNA or RNA of variable length which can be radioactively
labelled. It can then be used in DNA or RNA samples to detect the presence of
nucleotide sequences that are complementary to the sequence in the probe.

Motifs – These are short, recurring patterns in DNA that are presumed to have a
biological function. Often they indicate sequence-specific binding sites for
proteins such as nucleases and transcription factors (TF).
SNP – It is a variation in a single nucleotide that occurs at a specific position in
the genome, where each variation is present to some appreciable degree within a
population (Single Nucleotide Polymorphism)
Transposable DNA – The sequences of DNA that move (or jump) from one location
in the genome to another.
Repetitive DNA – These are patterns (also known as repetitive elements
or repeats) of nucleic acids (DNA or RNA) that occur in multiple copies throughout
the genome.
Selection pressure- An agent of differential mortality or fertility that tends to make
a population change genetically.

High throughput gene sequencing- High-throughput (formerly "next-generation")
sequencing, applies to genome sequencing, genome resequencing transcriptome
profiling (RNA-Seq). The high demand for low-cost sequencing has driven the
development of high-throughput sequencing technologies that parallelize the
sequencing process, producing thousands or millions of sequences concurrently

AIM
 Generation and analysis of genomic sequences of diverse bacterial,
oomycete, fungal, and viral pathogens
INTRODUCTION
 Identify genetic sources of virulence
 Understand differences observed among related pathogens
 Guide the development of diagnostic tools capable of discriminating
among specific strains
 Reveal sources of host resistance
 Understand the dynamics of host-microbe interactions and the
diseases they cause.
Source: https://pppmb.cals.cornell.edu
 Pathogen + Genomics = Pathogenomics

History of Genome Sequencing
Source: http://genomics.energy.gov/

TECHNIQUES INVOLVED
1. Marker Gene Analysis
 The targeted amplification and sequencing of taxonomically informative
‘marker genes’ from microbial community DNA is known as marker gene
analysis
 A genetic marker is a gene or DNA sequence with a known location on a
chromosome that can be used to identify individuals or species.
 Strength- Deep coverage allows characterization of rare species.
2. Metagenomics
 Study of genetic material recovered directly from environmental samples
 The broad field may also be referred to as environmental genomics, Eco
genomics or community genomics.
 Strength- Unbiased profiling, Allows genomic studies of uncultured microbial
species.

3. Meta-transcriptomic
 The extraction and analysis of metagenomic mRNA
 Provides information on the regulation and expression profiles of
complex communities as Metagenomics allows researchers to access the
functional and metabolic diversity of microbial communities, but it
cannot show which of these processes are active
 Strength- Transcript abundance can be determined
More sensitive for detecting alterations in gene
expression than proteomics
4. Meta-proteogenomics
 Study of active functionalities and pathways
 Strength- Better approximation of levels of functional activities with
proteomics than transcriptomics
Source: Guttman et al., 2014

Effectors
o Effectors are proteins expressed by plant pathogens to aid infection
of specific plant species
o Modular proteins with cleavable amino-terminal secretion signals.
o Known to disable extracellular and subcellular plant defenses
o Enable the pathogen to adapt to the protease-rich environment of
the plant
o In nutshell, pathogen effectors have proved to be useful probes to
identify plant proteases that have roles in immunity
Source: Pais et al., 2013

 ROLE OF EFFECTORS
 Pathogen effector proteins can act as virulence factors to suppress the basal
defense mechanisms.
 Effector genes are under dynamic, even opposite selection pressure depending
on the host plant genotype that the pathogen interacts with.
 Immune processes
 Useful tools for probing plant germplasm for new resistance traits and are
poised to improve the breeding and deployment of disease resistance genes in
agriculture.
 Due to the diverse and rapidly evolving effector gene complements P.
infestans and some other plant parasitic pathogens are very much successful in
causing disease.
o Host pathogen interaction
o Cellular dynamics

 Classes of Effectors
1. Apoplastic Effectors 2. Cytoplasmic Effectors
 Secreted into the plant
extracellular space
 Modular proteins with cleavable
amino terminal secretion signals
 Disables extracellular plant
defense and enable the pathogen
to adapt to the protease-rich
environment of the plant
apoplast
 Translocated inside the plant cell
 Carry an additional domain after
the signal peptide that mediates
translocation inside host cells and
is defined by conserved motifs
Eg. Serine and cysteine protease
inhibitors, phytotoxin-like SCR74.
Eg.RXLR, CRN and CHXC
Source: Kamoun, 2006

 EFFECTOR GENE EXPRESSION
 Highthrough put gene expression analysis by sequencing of cDNA
libraries, microarray and RNA-seq technologies are valuable tools to
identify candidate virulence factors
 Gene expression profiles in pathogen exhibits dynamic changes
throughout the different developmental stages of their life cycle,
dramatic changes occur during infection of host plants

 Expression patterns of oomycete effector genes

KEY MEANING
epi 1 (PITG_22681) Apoplastic effector that
functions as protease
inhibitor
Avr3a (PITG_14371) Cytoplasmic RXLR eff ector
Ubiquitin and pisp3 Controls
Dpi Days post-inoculation

 HOST PLANT RESPONSES TO EFFECTOR
 Plants respond to pathogen effectors in various ways depending on
the genotypes of both the plant and the invading pathogen
 Plant response to pathogen effectors- PAMP/AVR/ETI (Vleeshouwers
et al., 2008)
 Virulence activities of effector protein (Fabro et al.,2011)
 Discovery of new resistance genes and cloning of R genes
(Goritsching et al.,2012)

Dr.Y.S.ParmarUniversityofHorticulture&Forestry Pathogenomics with reference to Plant pathology
 Unraveling plant processes
 Surveys of pathogen’s genome sequence
 Development of diagnostic tools
 Durable resistance

 The activation of host immunity by effector proteins helps the dissection of
plant susceptibility and resistance mechanisms
 Unraveling plant processes
 Identification of host proteins that interact with pathogen effectors can give
insights into the plant pathways targeted and perturbed during the infection
process
 Effectors can be used as molecular probes to study the structural changes that
occur during plant infection at a subcellular level
 The suppression of plant immune responses under the influence of pathogen.
Eg. Production of reactive oxygen species (ROS)

Source: Pais et al.,2013
 High-throughput pipeline for using plant pathogen effectors to unravel
plant processes

Surveys of pathogen’s genome sequences
• Lifestyle-associated genomic adaptations- Necrotrophy/ Biotrophy/
Hemibiotrophy
• Diversity among pathogen. E.g. Oomycetes
 One striking feature of these oomycete genomes is the considerable
variability in size, ranging from 37 Mb for the biotrophic plant
pathogen Albugo laibachii (an obligate parasite of the model plant
Arabidopsis thaliana), to 240 Mb for the hemibiotrophic P. infestans
(which parasitizes tomato and potato)
 The genomes of necrotrophic plant fungi got an abundance of genes encoding
products that are intuitively associated with necrotrophic life
 Protein and carbohydrate hydrolases, are enzymes that accomplish the
degradation of the plant cuticle and cell wall

 These observed differences in genome size are largely due to the
proliferation of transposable elements and repetitive DNA, which in P.
infestans account for 74% of the genome content
 At 100 Mb, the downy mildew Hyaloperonospora arabidopsidis, an obligate
parasite of A. thaliana, also has a relatively large genome size, a recurrent
trend in biotrophic oomycete and fungal pathogens
 Repeat regions in these expanded genomes tend to be unstable; they may
promote genome duplication and shuffling, increased rates of mutagenesis and
gene silencing
CONT…

Genomic analysis of oomycetes

Development of diagnostic tools
E.g. Wheat rust
o Field pathogenomics, whether using RNA sequencing (Hubbard et al., 2015)
or genomic DNA-based approaches, will supplement phenotyping.
o The non-viable samples can be transported to, and sequenced by regional
technology centers, providing standardized information that allows
comparative global analysis and monitoring.
o Dense SNP panels will allow tracking of pathogen dispersal on a global scale
without the need to move live pathogen samples. Once genotypic variants
have been identified, phenotypic characterization can focus on
representative samples, optimizing the use of national resources.

o This will allow prediction of which pathotypes are a threat to particular
genotypes within specific geographic regions and provide an early-warning
system for crop vulnerabilities.
o Unprecedented amounts of information on the genetic structure of rust
populations will provide detailed insights into the selective forces driving
evolution of new pathotypes
CONT…

Durable resistance
 Durable disease resistance, remains effective over long period of time in
environments favourable for disease
 The re-sequencing of multiple pathogen isolates provides insights into
genome organization, plasticity and the presence of pathogenesis-related
genes.
 Sequencing of isolates from multiple geographical areas will reveal the
prevalence and distribution of known virulence genes as well as uncover new
virulence genes.
 Pathogenomics provides the data on host and pathogen needed for
deployment of effective resistance genes

Contd.
 Continual global monitoring of the pathogen will enable anticipatory strategies
that take into consideration the frequency and relative fitness costs of the
targeted virulence effectors.
 Effective combinations of resistance genes can be deployed as pyramids of
individual specificities tailored to each region.
 Continual surveillance of pathogen populations will allow the impact of
different resistance genes and strategies to be monitored.
 This will facilitate rapid responses when resistance breaks down and inform
temporal and spatial adjustments in resistance-gene deployment.

Xanthomonas
 Geographically and temporally diverse strains of Xanthomonas axonopodis pv.
manihotis (Xam), which is the causal agent of cassava bacterial blight was taken
for study.
 A phylogenetic analysis based on the genome data showed strong geographical
clustering of the strains, which indicates that local evolution is more important
than global gene transfer or strain migration.
 The distribution of T3SEs varied considerably among strains, but nine were
found to be present in all Xam isolates.
 This set of effectors are highly conserved over all geographical sampling sites
and 70 years of strain isolation.

 If these highly conserved T3SEs are essential because of their interaction with core
components of the cassava immune system, then they may provide insights into
the development of durable resistance to protect this important crop.
CONT…

Other methods for recovery of R genes
 Use of effectors in large-scale screens of germplasm has facilitated the
discovery of new resistance genes and their classification into discrete
recognition specificities, accelerating the cloning of R genes while avoiding
redundant cloning efforts
 Effectors can be used to identify R gene homologs in plant species that are more
compatible for breeding. Eg. Solanum stoloniferum and S papita for P infestans
RXLR effector .
 Monitoring of effector allele diversity in pathogen populations. This can provide
valuable information to assess the potential of a given R gene regarding its
spectrum and durability, and to design control strategies based on the dynamic
distribution of virulence alleles in a given population, allowing the early
detection of races that can overcome the deployed R genes.

CONTD…
 Genome and Transcriptome analysis can be used to determine the set of
effector genes present and expressed during infection by isolates of a given
genotype, providing information on the R genes that can be deployed in host
to control that particular genotype.
 By expanding the effector recognition specificity of a given R gene to new
virulent alleles by performing artificial evolution by random mutagenesis, an
approach that has been previously successful when applied to the PVX
resistance gene Rx .

Case study- 1

Materials and methods
 219 samples of wheat and triticale infected with PST from 17
different counties across the UK in the spring and summer of 2013
 Field pathogenomics reveals a shift in the PST population in the UK. It has
uncovered a dramatic shift in the PST population that could have serious
implications for wheat production in the UK
 Methodology described herein accelerates genetic analysis of pathogen
populations and circumvents the difficulties associated with obligate plant
pathogens

Result
 RNA-seq analysis of PST-infected plant material is a useful approach for
accurately genotyping isolates of PST directly from the field.
 Genetic diversity between members of a single population cluster for all
2013 PST field samples was much higher than that displayed by historical UK
isolates, revealing a more diverse population of PST.
Conclusion
 A dramatic shift in the PST population in the UK, likely due to a recent
introduction of a diverse set of exotic PST lineages
 In this study, we developed a robust and rapid method based on RNA
sequencing directly from infected host samples to gain insight into emerging
pathogen populations

Conclusion
 Next-generation sequencing technologies provide new opportunities
to study pathogens and the hosts they infect.
 High-throughput pathogenomics offers the possibility for analyzing a
large number of pathogen isolates and host varieties rapidly and at
low cost.
 It detects genes being expressed and therefore the determinants of
the interaction; thus, non-expressed genes present in the genome do
not obscure genotype-phenotype correlations.
 A potential limitation of the method is that it only samples at one
time-point and does not reveal the genetic potential of non-
expressed pathogen genes.

THANK- YOU
THANK-YOU

Pathogenomics

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