Project Title: VIGS mediated screening for identification of genes involved in dual abiotic (salt, draught) and biotic (Ralstonia solanacearum) stress tolerance in tomato.

Tomato is one of the most consumable vegetables in the world, and the limitation of its production is caused by bacterial wilt disease caused by Ralstonia solanacearum. Due to its devastating lethality, R. solanacearum is one of the most intensively studied phytopathogenic bacteria now, and the bacterial wilt disease of tomato is used as a model system for investigating mechanisms of pathogenesis.

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Objective:

Our research is focused on the response of combined stresses (salt and drought) with the R. solanacearum infection which can provide useful information to evaluate resistance of tomato against the bacterial wilt disease.

We, in present study, intend to identify the genes involved in dual abiotic (draught, salt) stress and pathogen (R. solanacearum) infection in tomato by utilizing VIGS based reverse genetics approach.

Methodology:

     In order to screen the genes involved in dual abiotic (draught, salt) stress and pathogen (R. solanacearum) infection in tomato, we shall initially subject the tomato plants to draught and salt stresses. In brief, the tomato plants will be subjected to following stress conditions along with R. solanacearum infection w.r.t control where no stress will be given:

(i)                 Salt stress (200 mM NaCl)

(ii)               Drought stress (150 mM PEG)

(iii)             Salt + draught stress

(iv)             No stress/ control

NOTE- Another Idea-

The cDNA Library can also be prepared from control along with three stresses:

a.       Control

b.      Stress 1- Salt

c.       Stress 2- Drought

d.      Stress 3- combined Salt and drought.

The upregulation and downregulation of various genes can be studied, and out of these the best upregulated genes w.r.t. control and other individual stress can be checked and then cloned in TRV vector followed by the test with Tomato plants along with R. solanacearum infection.

The selection of proper cDNA library can be prepared by comparing the control w.r.t. to combined stress and also abiotic stresses (Figure given below).

 

     Following the above-mentioned procedure, cDNA library will be prepared in TRV2 vector (cDNA library construction in TRV2 vector as described in by Liu and Page, 2008 will be followed). The TRV vector encoding the gene is first transformed in E. coli  host cell and screened with the VIGS sequence specific primer or adapter specific primers followed by transformation in Agrobacterium host cells.

 

     The cDNA clones from the stress library will be used to carry out VIGS mediated screening in tomato plants, and each positive colony will be used to identify the composition of that particular gene following sequencing procedures.

 

     For in planta characterization of the selected stress responsive tomato genes, the agro infiltration is done by infiltration on primary leaves of plants in a single pot using a syringe without a needle. The suspension will be infiltrated in abaxial surface of healthy tomato leaves of pot grown tomato plants (The suspension Agrobacterium cultures containing individual TRV cDNA library clones were each mixed with an equal volume of a culture of Agrobacterium expressing the TRV RNA 1 construct).

 

The negative control plants will be infiltrated with TRV1 and TRV2-empty vector. The plants will be allowed to grow for two weeks under controlled environmental conditions with proper nutrient supplementation because vigorous growth of plant is necessary for higher gene silencing.

 

     The R. solanacearum pathogen, which will be used to screen the resistant genes of tomato plants, will be designed to express GFPuv construct for easy detection of bacterial load by their inoculation in tomato leaves. The GFPuv protein fluoresce green in UV light.

 

     The R. solanacearum suspension expressing GFPuv protein will be infiltrated in the abaxial surface of the same leaves that were previously infiltrated with TRV1 + TRV2-empty vector (control leaves) or TRV1 + TRV2-cDNA insert (leaves where expression of respective tomato genes has to be silenced).

 

     Between 2-5 days, the leaves will be exposed to UV light to visually score the expression of GFPuv protein. The control leaves, where the resistant genes are functional are expected to have either no green fluorescence or little green fluorescence against the red florescence background of the leaves. On the other hand, if the concerned silenced gene is responsible for conferring resistance to Ralstonia solanacearum pathogen, the green fluorescence will be restored in the spot of leaves where the pathogen was inoculated.

     In this way, the resistant genes will be identified and functionally validated.

     The cDNA clones, which confer resistance against R. solanacearum pathogen, will be sequenced for their identification.

Future prospective of the proposed research:

The genes conferring tolerance to R. solanacearum pathogen, identified by cDNA library screening, can be overexpressed in tomato to develop abiotic stress and R. solanacearum tolerant transgenic tomato plants.

References:

Genin S, Denny TP (2012) Pathogenomics of the Ralstonia solanacearum species complex. Annu. Rev. Phytopathol. 50: 67-89.

Liu E, Page J (2008) Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus. Plant Methods. 4: 5, doi:10.1186/1746-4811-4-5.

Senthil-Kumar M, Mysore KS (2011) Virus-induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant Biotechnol. J. 9: 797–806.

Senthil-Kumar M, Lee HK, Mysore KS (2013) VIGS-mediated forward genetics screening for identification of genes involved in nonhost resistance. J. Vis. Exp. e51033.

Senthil-Kumar M, Ajith A, Uppalapati SR, Mysore KS (2008) Virus-induced gene silencing and its applications. CAB Rev. Perspect. Agri. Vet. Sci. Nutri. Nat. Res. 3: 1–18.