Influence of Host Chloroplast Proteins on Tobacco mosaic virus Accumulation and Intercellular Movement      

Abstract

Tobacco mosaic virus (TMV) forms dense cytoplasmic bodies containing replication-associated proteins (virus replication complexes [VRCs]) upon infection. To identify host proteins that interact with individual viral components of VRCs or VRCs in toto, we isolated viral replicase-and VRC-enriched fractions from TMV-infected Nicotiana tabacum plants. Two host proteins in enriched fractions, ATP-synthase g-subunit (AtpC) and Rubisco activase (RCA) were identified by matrix-assisted laserdesorption ionization time-of-flight mass spectrometry or liquid chromatography-tandem mass spectrometry. Through pulldown analysis, RCA bound predominantly to the region between the methyltransferase and helicase domains of the TMV replicase. Tobamovirus, but not Cucumber mosaic virus or Potato virus X, infection of N. tabacum plants resulted in 50% reductions in Rca and AtpC messenger RNA levels. To investigate the role of these host proteins in TMV accumulation and plant defense, we used a Tobacco rattle virus vector to silence these genes in Nicotiana benthamiana plants prior to challenge with TMV expressing green fluorescent protein. TMV-induced fluorescent lesions on Rca-or AtpC-silenced leaves were, respectively, similar or twice the size of those on leaves expressing these genes. Silencing Rca and AtpC did not influence the spread of Tomato bushy stunt virus and Potato virus X. In AtpC-and Rca-silenced leaves TMV accumulation and pathogenicity were greatly enhanced, suggesting a role of both host-encoded proteins in a defense response against TMV. In addition, silencing these host genes altered the phenotype of the TMV infection foci and VRCs, yielding foci with concentric fluorescent rings and dramatically more but smaller VRCs. The concentric rings occurred through renewed virus accumulation internal to the infection front.As obligate intracellular organisms, plant viruses must interact with various host factors essential for their accumulation and intercellular movement. These virus-host interactions are often complex and difficult to understand mechanistically, but for some of them considerable progress has been made in recent years (Ahlquist et al

Plant virus replication and movement

“…Silencing of GAPDH expression inhibited the accumulation of p35 in the cortical VRCs and also reduced the efficiency of virus movement thus indicating that the cortical VRCs are associated with RCNMV movement whereas the larger, perinuclear VRCs, that occur at a later time, rather function to maximize virus reproduction (Kaido et al, 2014). The role of GAPDH in facilitating the association of the p35 MP with the cortical VRCs adds to the potential role of chloroplastic proteins in virus replication as already noted by other studies (Bhat et al, 2013;Cheng et al, 2013;Lin et al, 2007). It remains to be seen whether the p35 MP associated with cortical VRCs targets the whole VRC or rather a VRC subcompartment to PD to spread infection.…”

A Violaxanthin Deepoxidase Interacts with a Viral Suppressor of RNA Silencing to Inhibit Virus Amplification

“…Recently, ATP-synthase g-subunit and Rubisco activase were reported to be isolated from the TMV replication complex. TMV infection down-regulated their mRNA levels, and silencing of them enhanced viral accumulation (Bhat et al, 2013). Therefore, ZmVDE likely plays a similar role in defense against viral infection.…”

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS

“…The inoculated maize plants were photographed at various dpi. Gene knockdown efficiency by the original or the modified BMV vectors was determined through qRT-PCR as described previously (Bhat et al, 2013) with specific modifications. Total RNA from pooled second and third systemically infected maize leaves was extracted and treated with DNase I enzyme as described in the section, "Virus Accumulation in N.…”