Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

Abstract

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-tocell movement (for review, see Benitez-Alfonso et al.,

Viral protein suppresses oxidative burst and salicylic acid‐dependent autophagy and facilitates bacterial growth on virus‐infected plants

“…It is worth noting that the TOR‐binding and dsR motif‐containing domain of CaMV P6 has also been reported to physically interact with the ribosomal proteins L13 and L18, both implicated in viral translation (Ryabova et al ., ). Furthermore, a larger mini‐TAV sequence containing the TOR‐binding domain physically interacts with the chloroplast outer membrane protein CHUP1 and the plasma membrane (plasmodesmata‐associated) C2‐calcium‐dependent protein AtSRC2.2, both implicated in viral movement in Arabidopsis (Angel et al ., ; Rodriguez et al ., ). Interestingly, a pepper homolog of AtSRC2.2 is also involved in plant defenses against viral and nonviral pathogens (Kim et al ., ).…”

“…P6 is a multifuctional protein essential for CaMV virulence, because it is a main component of the viroplasm (viral replication and assembly factory), a transactivator of pgRNA translation, and a suppressor of RNA silencing (Hohn, ; Hohn & Rothnie, ). P6 has also been implicated in viral movement (Harries et al ., ; Angel et al ., ; Rodriguez et al ., ). Moreover, P6 is a main determinant of the host range and pathogenicity and an Avr factor that triggers HR in resistant hosts (Daubert et al ., ; Schoelz et al ., ).…”

Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90

“…Studies have shown that virus infection down-regulates chloroplast-and photosynthesis-related proteins and genes (CPRPs and CPRGs, respectively) in virus-infected plants [11,15,22]. Rice stripe virus (RSV) downregulates CPRGs during infection and disturbs the photosynthesis [23].…”

Mutations within A 35 amino acid region of P6 influence self-association, inclusion body formation, and Caulimovirus infectivity

“…Alternatively, the inability to cleave the N-terminus of the capsid protein in small bodies might also be expected to inhibit the maturation of virions. A recent study indicated that P6 edIBs may have a role in delivery of virion to plasmodesmata (Rodriguez et al, 2014). Consequently, CaMV mutants trapped in the small body stage might be less efficient in movement of virions to adjacent cells.…”

“…P6 also interacts with CHUP1, a plant protein localized to the outer membrane of chloroplasts that is essential for chloroplast movement on microfilaments in response to changes in light intensity (Angel et al, 2013). The interaction of P6 with CHUP1 likely contributes to intracellular movement of CaMV for delivery of virions to the plasmodesmata (Rodriguez et al, 2014). …”