Host-delivered RNAi-mediated silencing of Phytophthora infestans for imparting late blight resistance in potato
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Date
2017
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DEPARTMENT OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE TECHNOLOGY AND SCIENCES, ALLAHABAD (U.P.) - INDIA 211007
Abstract
Potato (Solanum tuberosum L.) is currently, the third most consumed crop worldwide
after wheat and rice. Consumption of potato is expanding strongly in the developing
nations which account for more than half of the global harvest. Late blight of potato
caused by the oomycetes Phytophthora infestans, that destroys leaves, stems, and tubers,
is the decimator of potato cultivation costing over $ 13 billion annually all over the world
in crop losses and control measures. The strategies for managing potato late blight often
remain unsustainable and costly. The control measures for late blight mainly rely on
fungicides. In the present scenario, late blight resistant breeding still pose a great
challenge to potato breeders, due to the breakdown of most of the resistant varieties
developed earlier. RNAi is interesting mechanistically for reasons including its action at
the posttranscriptional level, its exquisite sequence selectivity and the flexibility it affords
for regulating gene expression. Silencing of the Avr3a gene through HD-RNAi in the
transgenic background could lead to pathogen death and/or loss of virulence, and
consequently imparting late blight resistance.
In the detached leaf assay, among the siRNA mediated silenced lines of Kufri
Khyati, least lesion area was recorded in the line K. Khyati 1129 (4.89 cm2) and K.
Khyati 1037 (4.97 cm2) in comparison to other lines and control whereas in Kufri
Pukhraj lines, it varied from 8.05 cm2 to 11.88 cm2 while in control it was 13.54 cm2.
LER values as well as spore count was also reduced in siRNA transformed lines as
compared to control plants. Whole plant bioassays against P. infestans revealed delayed
and moderate resistance at 120 hpi, with a resistant nature observed at 72 hpi in four
lines. Massive pathogen sporulation was observed on the wild-type in comparison to the
reduced sporulation observed on susceptible transgenic lines at 120 hpi. No sporulation
was observed in the moderate resistant lines of both cultivars. Transcript levels of Avr3a
were significantly increased (line 2086, 4.2-fold; line 2155, 10.4-fold; line 2173, 7-fold)
in leaf samples withdrawn at 0 hpi, and subsequent expression levels decreased to less
than 1-fold from 72 to 120 hpi in all selected partial resistant lines compared with nontransformed
controls. Pathogen DNA quantity increased progressively as the disease
symptoms increased sharply over time up to 120 hpi. The quantification assay also
revealed that partial resistant lines had less pathogen biomass compared with the nontransformed
controls. All plants showed low intensity siRNA expression before
inoculation, the expression was greatly increased in the transgenic lines (Kufri Pukhraj
2086 and 2173) compared with the control and susceptible transgenic lines not showing
siRNA expression. Southern blot analysis confirmed at least one T-DNA copy integrated
into the genome of the transgenic plants (Kufri Khyati 1037, Kufri Pukhraj 2173, single
copy; Kufri Pukhraj 2086, three copies; Kufri Pukhraj 2155, single copy); no integration
of the gene was observed in the control plants.
Description
Ph.d thesis
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