The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. Intercellular movement of plant viruses is the crucial step during systemic viral infections. Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus, forms movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum in infected cells, which are required for efficient intercellular movement of the virus. We provide evidence that these cortical punctate structures constitute the viral replication complex (VRC), which forms during the early stage of virus infection. Furthermore, we show that a host protein of Nicotiana benthamiana, glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), possibly intercalates between the cortical VRC and MP. Knockdown of NbGAPDH-A diffused subcellular localization of MP and reduced intercellular movement of the virus. Chloroplastic NbGAPDH-A relocalized to the cortical VRC after infection with the virus. Our results suggest that the cortical VRC serves not only as the replication factory of viral RNA but also as a transportation hub, which transports viral RNA to neighboring uninfected cells via plasmodesmata.

Robust nonhost resistance of Arabidopsis thaliana against the nonadapted hemibiotrophic fungus Colletotrichum tropicale requires PEN2-dependent preinvasive and CYP71A12/CYP71A13-dependent postinvasive resistance, which both rely on tryptophan (Trp) metabolism. Here we report that CYP71A12 and CYP71A13 are critical for Arabidopsis’ postinvasive resistance toward both the necrotrophic Alternaria brassicicola and the adapted hemibiotrophic C. higginsianum fungi. Metabolite analyses suggest that the production of indole-3-carboxylic acid derivatives (ICAs) and camalexin is induced upon pathogen invasion, while phenotypic comparison of cyp79B2 cyp79B3 and pen2 cyp71A12 cyp71A13 plants indicates that the contribution of ICAs to postinvasive resistance is dose-dependent. We also found that the disruption of intact pattern recognition receptor complex caused by bak1–5 mutation significantly reduced postinvasive resistance against C. tropicale and A. brassicicola , indicating that pattern recognition commonly contributes to this second defense-layer against pathogens with distinct infection strategies. However, the bak1–5 mutation had no detectable effects on Trp-metabolite accumulation triggered by pathogen invasion. Together with this, further comparative gene expression analyses suggested that pathogen invasion in Arabidopsis activates (i) bak1–5 insensitive Trp-metabolism that leads to antimicrobial secondary metabolites, and (ii) a bak1–5 sensitive immune pathway that activates the expression of antimicrobial proteins.

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5' end of RNA1. Stem-loop structures were predicted in the 5'-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5'-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5'-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus.