BackgroundViral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.Principal FindingsTomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.SignificanceDetails of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.

Plants have evolved diverse mechanism to recognize pathogen attack and triggers defense responses. These defense responses alter host cellular function regulated by endogenous, small, non-coding miRNAs. To understand the mechanism of miRNAs regulated cellular functions during stem rust infection in wheat, we investigated eight different miRNAs viz. miR159, miR164, miR167, miR171, miR444, miR408, miR1129 and miR1138, involved in three different independent cellular defense response to infection. The investigation reveals that at the initiation of disease, accumulation of miRNAs might be playing a key role in hypersensitive response (HR) from host, which diminishes at the maturation stage. This suggests a possible host-fungal synergistic relation leading to susceptibility. Differential expression of these miRNAs in presence and absence of R gene provides a probable explanation of miRNA regulated R gene mediated independent pathways.

We investigated viral gene suppression in an infected tomato, by transforming it with RNA inhibition (RNAi) constructs derived from same viral gene. To develop RNAi constructs, conserved sequences ranging from 21 to 200 nt of the viral target AC4 gene of various viruses causing the tomato leaf curl disease were chosen. The double-stranded (ds)RNA producing constructs carry the sense and antisense portions of these sequences and are separated by different introns behind a constitutive promoter. We compared the levels of suppression of the viral target gene by transforming four different RNAi constructs with varied arm length of dsRNA. Gene silencing levels of the viral target gene were found to be directly proportional to the arm length of the dsRNA. We observed that dsRNA derived from longer arm-length constructs generating a pool of siRNAs that were more effective in targeting gene silencing. After transformation, one of the RNAi construct having a 21 nt arm-length produced aberrant phenotypes. These phenotypic anomalies may be due to unintended ('off-target') host transcript silencing. The unintended host transcript silencing showed modest reversion in the presence of the viral target gene. The findings presented here suggest that the arm length of dsRNA capable of producing a pool of diced siRNAs is more efficient in gene silencing, the effect of off-targeting siRNA is minimized in a pool, and off-targeting silencing can be minimized in the presence of target gene.

We sequenced the genome of a strain of the Gram-negative bacterial species Elizabethkingia anophelis, which is an important component of the Anopheles mosquito microbiome. This genome sequence will add to the list of resources used to examine host-microbe interactions in mosquitoes.

The genome of Cucumber mosaic virus New Delhi strain (CMV-ND) from India, obtained from tomato, was completely sequenced and compared with full genome sequences of 14 known CMV strains from subgroups I and II, for their genetic diversity. Sequence analysis suggests CMV-ND shares maximum sequence identity at the nucleotide level with a CMV strain from Taiwan. Among all 15 strains of CMV, the encoded protein 2b is least conserved, whereas the coat protein (CP) is most conserved. Sequence identity values and phylogram results indicate that CMV-ND belongs to subgroup I. Based on the recombination detection program result, it appears that CMV is prone to recombination, and different RNA components of CMV-ND have evolved differently. Recombinational analysis of all 15 CMV strains detected maximum recombination breakpoints in RNA2; CP showed the least recombination sites.