Molecular analysis of multi-layered mechanisms of non-host resistance in Arabidopsis thaliana to bromoviruses

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Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. BMV (M1 strain) shows limited infection in a model plant Arabidopsis thaliana, while SBLV efficiently infects A.thaliana. Even in protoplasts, accumulation levels of BMV were lower than those of SBLV and were enhanced by the cpr5 mutations. Efficiency of translation mediated by the 5' and 3' noncoding regions of BMV RNAs 1 and 2 that encode replication proteins 1a and 2a, respectively, was enhanced in cpr5-2 mutant. This suggests that at least enhanced translation of viral components associated with replication allow BMV to efficiently infect cpr5 mutants. In contrast to the M1 strain of BMV, the M2 strain of BMV efficiently multiplied in A.thaliana protoplasts without cpr5 mutations, while they showed similar accumulations in barley protoplasts, a systemic host of BMV. Northern blot analyses using reassortants and hybrid viruses between BMV-M1 and -M2 demonstrated that sequences within the tRNA-like structure at the 3' end of BMV-M2 RNA2 most remarkably contributed to the efficient multiplication of BMV-M2 in A.thaliana cells. Luciferase reporter assay showed that the noncoding regions of M2 RNA2 enhanced translation more than those of M1 RNA2 in A.thaliana cells but similarly in barley cells, suggesting that the high level expression of 2a protein from RNA2 supported the efficient multiplication of BMV-M2 in A.thaliana cells. We have cloned the NbNACa1 gene from a model plant Nicotiana benthamiana that encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms and interacted with BMV movement protein in vivo. Downregulation of the NbNACa1 gene reduced cell-to-cell movement of BMV in N.benthamiana. Immunofluorescence microscopy observation suggests that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.