Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from

Trichoderma virens

Presentation by: Kalyani Rajlaingham

Introduction

Are transgenic plants protected against R. Solani (fungus)?

R. Solani is very susceptible to chitinase activity

Fungus cell wall consists of chitin

Chitinase genes from Trichoderma harzianum used to confer resistance to crop plants

Purpose

“The present study was undertaken to examine the effectiveness of the 42 kDa endochitinase genes from T. virens in protecting cotton from fungal diseases. In addition, tobacco plants were initially transformed to test the expression of various endochitinase clones, and then these were evaluated for their resistance to A. alternata. “

Materials andMethods

Step 1 – Choose genes, and deliver it to plant genome

Step 2 – Verify incorporation into genome. Copy Number?

Step 3 – Protein levels – How much protein does it make?

Step 4 – Treatments

Materials and Methods

Step 1 – Choose genes –

Tv-ech1, Tv-ech2, Tv-ech3 (cDNA clones)Tv-ech1g (genomic clone)

Materials and Methods Step 1 – Deliver it to the plant

genome –

Materials and Methods Step 1 – Deliver it to plant

genome –

Materials and Methods

In your hands, you now possess a transgenic plant.

Materials and Methods Step 2 – Verify incorporation into

genome. Copy Number?

Materials and Methods

Step 3 – Protein levels using Fluorometric gel-based

endochitinase assay

4-methylumbelliferyl B-D-N-N,N-

triacetylchitotrioside

Materials and Methods Step 4 – Test effectiveness of

insert in COTTON using the fungus R. Solani

Measure disease symptoms

Materials and Methods Step 4 – Test effectiveness of

insert IN TOBACCO using the fungus A. Alternata

% necrosis on leaf after 2 weeks was determined

A. Alternata

One ot two agar plugs

Results

Step 1 – Check that the transgene was inserted. In how many lines?

Step 2 – How many copies inserted?

Step 3 – Protein levels?

Step 4 – Present in subsequent generations?

Step 5 – Present in leaves, and roots?

Step 6 – Is transgenic plant resistant to R. Solani, and A. Alternata?

Results Step 1 – Inserted in

how many lines?

Results Step 1 – In Tv-ech1, endochitinase activity?

Results Step 1 –

Check that the protein – 42kDA- is present

Results Step 2 – How many

copies inserted?Southern Blot Northern Blot

ResultsStep 3 – Protein

levels? In Cotton

ResultsStep 3 – Protein

levels? In Tobacco

Results

Step 3 – Protein levels?

Results Step 6 –

Is transgenic Cotton resistant to R. Solani?

1 Week

Results

Step 6 – Is Cotton resistant to R. Solani?

2 Weeks

Results Step 6 – Is Tobacco resistant

to A. Alternata?

One Agar Plug

Results

Step 6 – Is Cotton resistant to

A. Alternata? (a)Two Agar Plugs

(b)One Agar Plug

Discussion

1 - No morphological abnormalities in transgenic plants

2 - Transgenic seedlings are resistant to R. solani, and A. Alternata

EXTRA

Discussion

1 - only one (Tv-ech1) of the three Trichoderma virens endochitinase cDNA clones tested as determined by the enzyme activity assays on leaf extracts

2 – no morphological abnormalities in transgenic plants

3 - high expressing lines identified in T0 generation were not always found to maintain chitinase activity in the T1 generation

Discussion

4 - T2 seeds from several high endochitinase-expressing lines were subjected to infection by planting them in soil infested with R. Solani

5 - At moderate inoculum pressure (0.28 g culture /L of mix), a majority of the untransformed seedlings (98%) in infested soil died due to post-emergence infections or the seeds failing to germinate; however, > 67% of the transgenic seedlings remained healthy even after 2 weeks in the infested mix

Discussion

6 - when the inoculum pressure was doubled (0.56 g /L), 15 of the 28 transgenic seedlings examined 9 days after planting were free of disease symptoms, while none of the control plants survived

Discussion

7 - A possible explanation for the high levels of protection observed in our study may therefore be that transgenic Trichoderma endochitinase activity in plant tissues may release compounds from the cell wall of the invading fungi, that in turn elicit in the plant a faster and more comprehensive defensive response.