Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens By Kalyani Rajalingham

1. Enhanced fungal resistance in
transgenic cotton expressing an
endochitinase gene from
Trichoderma virens
Presentation by: Kalyani Rajlaingham

2. Introduction

3. Are transgenic plants protected against R. Solani
(fungus)?
R. Solani is very susceptible to chitinase activity

4. Fungus cell wall consists of
chitin

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

6. 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. “

7. Materials and
Methods
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

8. Materials and
Methods
Step 1 – Choose genes –
Tv-ech1, Tv-ech2, Tv-ech3 (cDNA
clones)
Tv-ech1g (genomic clone)

9. Materials and
Methods Step 1 – Deliver it to the plant
genome –

10. Materials and
Methods Step 1 – Deliver it to plant genome
–

11. Materials and
Methods
In your hands, you now possess a
transgenic plant.

12. Materials and
Methods Step 2 – Verify incorporation into
genome. Copy Number?

13. Materials and
Methods
Step 3 – Protein levels using
Fluorometric gel-based endochitinase
assay
4-methylumbelliferyl B-D-
N-N,N-triacetylchitotrioside

14. Materials and
Methods Step 4 – Test effectiveness of insert in
COTTON using the fungus R. Solani
Measure disease symptoms

15. Materials and
Methods Step 4 – Test effectiveness of insert IN
TOBACCO using the fungus A. Alternata
% necrosis on leaf after 2 weeks was
A. Alternata
One ot two
agar plugs

16. 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?

17. Results
Step 1 – Inserted in how
many lines?

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

19. Results
Step 1 –
Check that the protein – 42kDA- is present

20. Results
Step 2 – How many
copies inserted?
Southern Blot Northern Blot

21. Results
Step 3 – Protein levels? In
Cotton

22. Results
Step 3 – Protein levels? In
Tobacco

23. Results
Step 3 –
Protein
levels?

24. Results Step 6 –
Is transgenic Cotton
resistant to R. Solani?
1 Week

25. Results
Step 6 –
Is Cotton resistant
to R. Solani?
2 Weeks

26. Results Step 6 – Is Tobacco resistant to
A. Alternata?
One Agar Plug

27. Results
Step 6 –
Is Cotton resistant to A.
Alternata?
(a)Two Agar Plugs
(b)One Agar Plug

28. Discussion
1 - No morphological abnormalities in
transgenic plants
2 - Transgenic seedlings are resistant to R.
solani, and A. Alternata

30. EXTRA

31. 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

32. 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

33. 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

34. 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.