TRANSCRIPT PROFILING IN HOST TRANSCRIPT PROFILING IN HOST PATHOGEN INTERACTIONPATHOGEN INTERACTION

Chairman : Dr.K.Prabakar(Professor)

Student : Mohammed Faisal P (09-614-007)

Topics coveredTopics covered

Transcription

Methods of transcript profiling

Host Vs Non host resistance

Basal defence

Gene expression analysis

Plant pathogen interaction

IntroductionIntroduction

Transcription is the process of creating an equivalent RNA copy of a sequence of DNA

Transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA and other non coding RNA produced in one or a population of cells

Transcriptomics is the branch of molecular biology that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type.

Cont.,

During transcription, a DNA sequence is read by RNA polymerase which produces a complementary, antiparallel RNA strand

Transcription is the first step leading to gene expression

Cont.,

“Central Dogma” of Plant Pathology

Bent and Mackey, 2007

MethodsMethods

• Northern Blotting

• EMSA

• DNA microarray

• SAGE Analysis

Northern BlottingNorthern Blotting

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample

Used for studying cellular control over structure and function by determining the particular gene expression

The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford

(Alberts et al, 2001)

Cont.,

EMSAEMSA

• A mobility shift assay is electrophoretic seperation of a protein-DNA or protein-RNA mixture on a polyacrylamide or agarose gel for a short period (about 1.5-2 hr for a 15- to 20-cm gel).

• The speed at which different molecules (and combinations thereof) move through the gel is determined by their size and charge, and to a lesser extent, their shape.

(Ausubel and Frederick, 2008)

Cont.,• The control lane will contain a single band corresponding to the unbound DNA or RNA fragment.

• If protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger,

• Less mobile complex of nucleic acid probe bound to protein which is 'shifted' up on the gel (since it has moved more slowly).

DNA microarrayDNA microarray

• A multiplex technology

• Arrayed series of thousands of microscopic spots of DNA oligonucleotides,

(Streit et al., 2007)

SAGESAGE

Serial analysis of gene expression (SAGE) 

  Used  to  produce  a  snapshot  of the mRNA population  in  a  sample  of interest  in  the  form  of  small  tags  that correspond  to  fragments  of  those transcripts

HOST VS NON HOST – PATHOGEN HOST VS NON HOST – PATHOGEN

INTERACTIONINTERACTION Difference between host species and nonhosts is haustorium formation stage in case of fungal pathogen

In nonhost tissues, the infection stops by cell wall-associated defense responses before any mature haustorium could appear.

Defense responses in resistant species were triggered when haustoria were fully visible and corresponded to hypersensitive responses

(Diez et al., 2008)

Interaction between Interaction between ArabidopsisArabidopsis and and P. P. infestansinfestans

Interaction between Interaction between ArabidopsisArabidopsis and and P. P. infestansinfestans

Hypersensitive response (HR) in Arabidopsis, 46 h after inoculationwith Phytophthora infestans.

(Edger (Edger et alet al., 2003 )., 2003 )

(Edger et al., 2003 )

Induction of Arabidopsis (A) PR1::GUS and (B) BGL2::GUS expression by Phytophthora infestans.

Cont.,Cont.,

XopD, a type III effector from Xanthomonas campestris pv vesicatoria (Xcv).

XopD effector (545 amino acids) encodes a Cys protease that cleaves tomato small ubiquitin-related modifier (SUMO).

XopD- delay tissue collapse with reduced salicylic acid (SA) levels, and modulation of host mRNA levels encoding senescence- and defense-associated genes.

CONT..

Consists of DNA binding domains, and two conserved transcriptional repressor motifs.

XopD is a unique virulence factor that promotes Xcv growth and suppresses host defense and pathogen-induced cell death responses triggered in diseased tissue.

XopD Is Required for Maximal Xcv Growth and to Delay theDevelopment of Disease Symptoms in Infected Tomato

Leaves.

XopD Suppresses Senescence-Associated XopD Suppresses Senescence-Associated Responses during Infection.Responses during Infection.

XopD Has Two EAR Motifs Required for Virulence in TomatoXopD Has Two EAR Motifs Required for Virulence in Tomatoand Tissue Necrosis in and Tissue Necrosis in N. benthamianaN. benthamiana

Differntial Expression of genes in rice against Differntial Expression of genes in rice against XooXoo Vs Vs XocXoc

(Nino et al., 2008)

“The general immune response to pathogens and other environmental stresses”

BASAL DEFENSEBASAL DEFENSEBASAL DEFENSEBASAL DEFENSE

(Ciardi et al., 2000)

Transgenic NahG potato plants unable to accumulate salicylic acid

Real time analysis showed enhancement of pathogen growth.

NahG plants pretreated with the salicylic acid analog 2,6- dichloro-isonicotinic acid showed same as wild plants

Salicylic Acid as signalling molecule for basal defence

Accumulation of A, salicylic acid (SA) and B, salicylic acid glucoside (SAG)

Phenotype and B, diameter of disease lesions 3 days after infection of wild-type (wt) and transgenic NahG plants (lines A and D2)

Microscopic analyses of wild-type (wt) and NahG plants after infection

Northern analysis of PR1A genes in transgenic and wild potato

GENE EXPRESSION

Transfer of DNA into a product

Acinetobacter sp was isolated from papaya dieback disease that caused dark spot at fruit surface. This will lead to rupture of the epidermis, which significantly favors pathogen spreading and disease dissemination

Cont.,

Down regulation of genes

Upregulation of gene

Plant – Fungal interactionsPlant – Fungal interactions

Biotrophic fungi and oomycetes, which require a living host for nutrients, must avoid recognition and activation of host defenses until they can complete their life cycles

Resistance gene leads to rapid recognition results in defense responses that inhibit further pathogen ingress

Many genes that are induced during defense responses are also induced during compatibility, with different kinetics

(Caldo Caldo et al.,et al., 2006) 2006)

Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays.

Use of a fluorescent protein-expressing fungus structure.

The LM-derived RNA, was of sufficient quality and quantity for global expression profiling using a fungal microarray.

A transgenic C. graminicola strain (Cgr-ASR-8#9a) expressing AmCyan protein driven by the strong constitutive RP27 promoter is used

Colletotrichum graminicola growth pattern in the maize stalk

Microarray analysis

The immune system’s role as defense is the ability to distinguish between ‘‘self’’ and ‘‘non-self’’.

This discrimination is partially achieved by the recognition of an invader’s chemical motifs by host surface receptors.

Potential pathogens have adapted numerous ways to overcome host immune systems

Plant – Bacterial interactionsPlant – Bacterial interactions

(Abramovitch et al., 2006 )

• Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive bacteria, causing wilt and canker disease in tomato

• Host responses to gram-positive bacteria and development of disease symptoms caused by Cmm in tomato are largely unexplored

• Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response

Inoculation and Regulation Inoculation and Regulation

Functional categories of Gene Expression

Wilting and Ethylene emission

EPILOGUEEPILOGUE