Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

Abstract

The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

The Jatropha leaf curl Gujarat virus on infection regulates the sugar and tricarboxylic acid cycle metabolic pathways in Jatropha

“…4). The similar negative regulation of sugar metabolism in tobacco plants due to JLCuGV infection was also reported in our earlier study [15]. Impaired sugar transport may be the reason for the increased sugar content during virus infection [25].…”

“…The Jatropha plants of different accessions like CP9, PCM, Ranpur, Shyamalaji, and Zanjmer were infected by geminivirus at Nesvad, Chorvadla and Bhadraval eld sites of CSIR-CSMCRI with >70% geminivirus disease incidence [15]. The symptoms were similar in all accessions with severe leaf curling.…”

“…The isolated DNA (100 ng) then used as template for detecting the presence of begomovirus by PCR using primers VirLCF (5'-GACCGCATAAATAATTGTGTGGGCCG-3') and VirLCR (5'-GCTGTAGAAATATATGCGTATTTTC-3'), which were designed speci cally for detecting the begomoviruses infecting Jatropha. The complete DNA-A of JLCuGV cloned in pBluescriptII KS(+) vector from our earlier study [15] was taken as a positive control.…”

“…1C). The virus responsible for causing the leaf curl disease was identi ed as JLCuGV in our earlier study [15]. The collected Jatropha leaf samples (three replicates each of healthy and infected leaf tissue) were checked for geminivirus infection.…”

“…The comparative metabolic pro les of an infected and healthy plant can help to unravel the changes in biochemical pathways on geminivirus infection. Earlier, we had identi ed the Jatropha leaf curl Gujarat virus (JLCuGV) infecting the Jatropha from Gujarat [15]. In the present study, the modi cations in metabolite pro ling were analysed to understand the regulation of the metabolic pathways on natural JLCuGV infection in Jatropha.…”