Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma...
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Transcript of Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma...
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.