Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.

Virus-infected plants often display developmental abnormalities that include stunting, leaf curling, and the loss of apical dominance. In this study, the helicase domain of the Tobacco mosaic virus (TMV) 126-and/or 183-kDa replicase protein(s) was found to interact with the Arabidopsis Aux/IAA protein PAP1 (also named IAA26), a putative regulator of auxin response genes involved in plant development. To investigate the role of this interaction in the display of symptoms, a TMV mutant defective in the PAP1 interaction was identified. This mutant replicated and moved normally in Arabidopsis but induced attenuated developmental symptoms. Additionally, transgenic plants in which the accumulation of PAP1 mRNA was silenced exhibit symptoms like those of virus-infected plants. In uninfected tissues, ectopically expressed PAP1 accumulated and localized to the nucleus. However, in TMV-infected tissues, PAP1 failed to accumulate to significant levels and did not localize to the nucleus, suggesting that interaction with the TMV replicase protein disrupts PAP1 localization. The consequences of this interaction would affect PAP1's putative function as a transcriptional regulator of auxin response genes. This is supported by gene expression data indicating that ϳ30% of the Arabidopsis genes displaying transcriptional alterations in response to TMV contain multiple auxin response promoter elements. Combined, these data indicate that the TMV replicase protein interferes with the plant's auxin response system to induce specific disease symptoms.

An interaction between the helicase domain of the Tobacco mosaic virus (TMV) 126-/183-kDa replicase protein(s) and the Arabidopsis thaliana NAC domain transcription factor ATAF2 was identified via yeast two-hybrid and in planta immunoprecipitation assays. ATAF2 is transcriptionally induced in response to TMV infection, and its overexpression significantly reduces virus accumulation. Proteasome inhibition studies suggest that ATAF2 is targeted for degradation during virus infection. The transcriptional activity of known defense-associated marker genes PR1, PR2, and PDF1.2 significantly increase within transgenic plants overexpressing ATAF2. In contrast, these marker genes have reduced transcript levels in ATAF2 knockout or repressor plant lines. Thus, ATAF2 appears to function in the regulation of host basal defense responses. In response to TMV infections, ATAF2 and PR1 display increased transcript accumulations in inoculated tissues but not in systemically infected tissues. ATAF2 and PR1 transcript levels also increase in response to salicylic acid treatment. However, the salicylic acid treatment of systemically infected tissues did not produce a similar increase in either ATAF2 or PR1 transcripts, suggesting that host defense responses are attenuated during systemic virus invasion. Similarly, noninfected ATAF2 knockout or ATAF2 repressor lines display reduced levels of PR1 transcripts when treated with salicylic acid. Taken together, these findings suggest that the replicase-ATAF2 interaction suppresses basal host defenses as a means to promote systemic virus accumulation.

The Tobacco mosaic virus (TMV) 126-kDa and read-through 183-kDa replicase-associated proteins have been shown to interact [Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T., Ishihama, A. (1999). J. Virol. 73, 2633-2640]. To identify and investigate the sequence required for this interaction, five segments covering different portions of the 126/183-kDa open reading frame, including the methyl-transferase, intervening region (IR), helicase-like (HEL), and polymerase domains, were screened via the yeast two-hybrid system against a library of TMV protein segments. Only one specific interaction between the HEL domain clone and a TMV library clone, IRnHEL, encoding the C-terminal half of the IR and the N-terminal portion of the HEL domain was identified. Sequence and deletion analysis revealed that the interacting clones share a region containing the helicase NTP-binding motif and that this region was essential for the interaction. To determine the functional significance of this interaction, mutants of the HEL domain segment that conferred a temperature-sensitive (ts) defect in the yeast interaction were identified and cloned into a recombinant TMV strain. Of the five selected mutants, three (V823I/S824N/V1042M, A877V, V1087I) produced a ts replication phenotype in protoplasts while the other two (A1073V, T884I) abolished TMV replication at both the permissive and the nonpermissive temperatures. An additional mutation, K839S, designed to disrupt the shared NTP-binding motif, nearly abolished the two-hybrid interaction and prevented virus replication, suggesting that NTP-binding and/or the structure of this motif is a contributing factor in the interaction. Taken together, these results provide support for an interaction between TMV replicase-associated proteins that involves specific structural features of the HEL and IR domains.

A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126-and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126-and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.Positive-stranded RNA viruses are a diverse group of pathogens that cause diseases in humans, plants, and animals. Although this group of pathogens is taxonomically diverse, they all encode replicase proteins involved in the synthesis of viral RNA. Enzymatic motifs within these replicase proteins can include methyltransferase (MT), helicase, and RNA-dependent RNA polymerase (POL) activities. These motifs may be present within a single multidomain protein, as found within the Tobacco mosaic virus (TMV) 183-kDa protein, or separated onto two or more virus-encoded proteins, as found in the 1a MT-HEL and 2a POL proteins of Brome mosaic virus (BMV) (2, 38). In infected cells, viral replicase proteins associate with host proteins as well as cellular membranes to produce replicase complexes that function in viral RNA synthesis. Despite the essential role of these replicase complexes in virus replication, little is known about their structure and the mechanisms that control their assembly.TMV is a positive-stranded RNA virus that has served as a model for the study of RNA replication (3). TMV is the type member of the genus Tobamovirus and a member of the alphavirus supergroup. Its genome encodes at least four proteins (11) (Fig. 1). The 5Ј-proximal open reading frames (ORFs) encode 126-and 183-kDa proteins, the larger produced by the read-through of an amber stop codon (33). Both the 126-and 183-kDa proteins are necessary for efficient virus replication (17,18,25). Homology studies indicate that the 126-kDa-protein-encoding ORF encodes MT and helicase domains divided by an uncharacterized intervening region (IR), while the readthrough portion of the 183-kDa-protein-encoding ORF encodes the POL domain (20,21,22) (Fig. 1). A 30-kDa cell-tocell movement protein and a 17.5-kDa coat protein are produced from 3Ј coterminal subgenomic mRNAs (6,16,28).Biochemical charac...