Sameer P. Goregaoker's research while affiliated with University of Maryland, College Park and other places

An interaction between the Tobacco mosaic virus (TMV) 126kDa replication protein and a host-encoded Rab GDP dissociation inhibitor (GDI2) was identified and investigated for its role in infection. GDI proteins are essential components of vesicle trafficking pathways. TMV infection alters the localization of GDI2 from the cytoplasm to ER-associated complexes. Partial silencing of GDI2 results in significant increases in the number of TMV infection foci observed in inoculated tissues. However, GDI2 silencing does not affect TMV accumulation at the infection site, cell-to-cell movement, or susceptibility of the host to mechanical inoculation. Furthermore, increases in the number of successful infection foci were specific to TMV and correlated with the appearance of vesicle-like rearrangements in the vacuolar membrane. Tissue infiltrations with brefeldin A, an inhibitor of vesicle trafficking, also enhanced host susceptibility to TMV. Combined these findings suggest that the 126kDa-GDI2 interaction alters vesicle trafficking to enhance the establishment of an infection.

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.

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 ∼30% 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.

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.

A transient expression system using onion epidermal cells was used to investigate domains of the Tobacco mosaic virus (TMV) 126-kDa replicase protein involved in cellular localization. Initially, a nuclear localization signal (NLS), identified within the amino-terminus of the 126-kDa protein, was investigated for its functionality using fusion constructs containing the green fluorescent protein (GFP). Fusion of the amino-terminal 70 amino acids of the 126-kDa protein, containing the NLS, to a beta-glucuronidase-GFP open reading frame (ORF), directed the accumulation of fluorescence to the nucleus. In contrast, similar constructs lacking the NLS or containing a mutated NLS sequence failed to accumulate within the nucleus. Additional investigations using GFP fusion constructs containing the first 178 or 388 amino acids of the 126-kDa protein also displayed nuclear localization. However, fusion constructs encoding the first 781 amino acids or the entire 126-kDa ORF did not accumulate within the nucleus but instead associated with the endoplasmic reticulum (ER), forming spot-like inclusions. Thus, a dominant ER association domain exists between amino acids 388 and 781 of the 126-kDa protein. Interestingly, a full-length 126-kDa GFP fusion construct encoding a nonfunctional NLS mutation also localized to the ER but did not form inclusions. Furthermore, a TMV mutant containing the same nonfunctional NLS mutation failed to replicate in protoplasts. Together these findings suggest that both the NLS and the ER retention domain contribute to the functional localization of the 126-kDa protein.

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.

Specific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549–557]. PVX-infected plants were subsequently challenge inoculated with 10 μg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene.