Coordinated Action of RTBV and RTSV Proteins Suppress Host RNA Silencing Machinery

Abstract

RNA silencing is as an adaptive immune response in plants that limits the accumulation or spread of invading viruses. Successful virus infection entails countering the RNA silencing machinery for efficient replication and systemic spread in the host. The viruses encode proteins with the ability to suppress or block the host silencing mechanism, resulting in severe pathogenic symptoms and diseases. Tungro is a viral disease caused by a complex of two viruses and it provides an excellent system to understand the host and virus interactions during infection. It is known that Rice tungro bacilliform virus (RTBV) is the major determinant of the disease while Rice tungro spherical virus (RTSV) accentuates the symptoms. This study brings to focus the important role of RTBV ORF-IV in disease manifestation, by acting as both the victim and silencer of the RNA silencing pathway. The ORF-IV is a weak suppressor of the S-PTGS or stable silencing, but its suppression activity is augmented in the presence of specific RTSV proteins. Among these, RTBV ORF-IV and RTSV CP3 proteins interact with each other. This interaction may lead to the suppression of localized silencing as well as the spread of silencing in the host plants. The findings present a probable mechanistic glimpse of the requirement of the two viruses in enhancing tungro disease.

Keywords: RNA silencing; RTBV; RTSV; coat protein; suppressors; virus disease.

Figure 1

Size distribution and mapping of small RNA reads obtained from tungro infected data sets: (A) Length distribution of the small RNA sequences showing the distribution of the first nucleotide in the molecules. (B) Plot of siRNA molecules mapping of ORF-IV for RTBV. The nucleotide position of ORF is shown on the X-axis. siRNAs mapping to the positive strand of the ORF are represented by blue lines, while those mapping to the negative strand are represented by red lines.

Figure 2

Reversal of GFP silencing by RTBV ORF-IV and its molecular analysis. (A) The different panels exhibit GFP fluorescence in the region infiltrated with ORF-IV at 3, 5, and 7 days post infiltration (dpi). The region infiltrated with the empty vector (EV) served as the control. (B) The graph represents the normalized Integrated Density Values (IDV) for the GFP and ORF-IV transcripts relative to 18S control cDNA at 3 dpi (d3), 5 dpi (d5), and 7 dpi (d7). (C) Northern blots of GFP transcript and GFP sRNA in regions infiltrated with ORF-IV (OIV) and EV. The 18S cDNA band served as the control. (D) The graph represents the normalized values of GFP transcripts and GFP siRNA as measured in the infiltrated regions at 3 dpi and 5 dpi. **, *** Significance at probability levels of 1% and 0.1%, respectively (ANOVA single factor).

Figure 3

Reversal of GFP silencing by co-infiltration of RTBV and RTSV ORFs. (A) The different panels exhibit representative pictures to show GFP fluorescence in the infiltrated regions at 3, 5, and 7 days post infiltration (dpi). The markings indicate the region infiltrated with (1) ORF-IV construct, (2) ORF-IV co-infiltrated with RTBV-ORF-I, (3) ORF-IV co-infiltrated with RTBV ORF-II, (4) ORF-IV co-infiltrated with RTSV ORF coding for coat protein 3, (5) ORF-IV co-infiltrated with RTSV ORF coding for protease, and (6) ORF-IV co-infiltrated with RTSV ORF coding for polymerase. (B) Normalized values for GFP transcripts in the different infiltrated regions at 5 dpi, confirmed using RT PCR. *, ** Significance at probability levels of 5% and 1%, respectively (ANOVA single factor).

Figure 4

Yeast two-hybrid assay for direct interaction study. Yeast colonies of pGAD-CP3 and pGBD-ORF-IV were co-transformed and selected on (i) two drop out plate (Leu⁻ Trp⁻), (ii) triple drop out (Leu⁻ Trp⁻ His⁻), and (iii) triple drop out (Leu⁻ Trp⁻ His⁻) supplemented with 5 μ M 3-AT. Co-infiltration of pGAD-CP3 with pGBD and pGAD with pGBD-ORF-IV was performed as the control.