Comparison of the Role of 5′ Terminal Sequences of Alfalfa Mosaic Virus RNAs 1, 2, and 3 in Viral RNA Replication

AbstractThe 5′ untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5′Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5′ UTR. When the 5′ UTR of RNA 3 was replaced by the 5′ UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5′ UTR of this RNA was replaced by 5′ terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5′ 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5′ 79 nt. However, when the 5′ 22 nucleotides of RNA 3 were replaced by the complete 5′ UTR of RNA 1 or 5′ sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5′ viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80–345 of the 5′ UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5′ end of the RNA

A Novel WRKY Transcription Factor Is Required for Induction of PR-1a Gene Expression by Salicylic Acid and Bacterial Elicitors[C][W]

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions −564 (box WK1) and −859 (box WK2). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK1 box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoter∷β-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35S∷NtWRKY12 and PR-1a∷GUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter