The etiology of disease remains one of the least understood areas of virology. Of particular importance is the identification of host components and pathways that directly interact with or are affected by the infecting virus. In this study, domains from the TMV replicase protein were assessed for their ability to interact with a library of Arabidopsis
host proteins. The virus replicase protein was selected for this study, because it is an essential component of the infection process and previously has been implicated in disease development (5
). Of the five replicase segments used to screen for interacting Arabidopsis
proteins, only the segment covering the TMV helicase domain yielded putative interacting clones. Of these cDNA clones, only those encoding the PAP1 ORF displayed levels of β-galactosidase activity indicative of a strong protein-protein interaction. In vitro interaction assays established the ability of the full-length viral replicase protein to interact with PAP1. Additional genetic and localization studies demonstrated a role for this interaction in disrupting the nuclear localization of PAP1. Specifically, the reduced ability of TMV-V1087I to interact with PAP1 corresponded with reduced disruptions in PAP1 localization and attenuated disease symptoms. Correspondingly, RNAi disruption of PAP1 produced a plant phenotype with similarities to the virus-induced disease response. Combined, these findings indicate that the interaction of the TMV replicase with PAP1 modulates the display of disease symptoms.
PAP1 encodes a 30-kDa member of the Aux/IAA family of early auxin-responsive proteins. PAP1, like other Aux/IAA proteins, contains four conserved domains involved in nuclear localization (domains I and II), protein destabilization (domain II), and dimerization (domains III and IV) (36
). At this time, the model for auxin signaling suggests that in the absence of auxin, Aux/IAA proteins form heterodimers with ARF proteins and repress their ability to modulate auxin response genes. In the presence of auxin, Aux/IAA proteins dissociate from ARF proteins and are targeted for degradation via the Skp1/Cullin/F-box subunit-containing E3 ubiquitin ligase complex, SCFTIR1
encodes the F-box component of this complex and interacts directly with Aux/IAA proteins to promote their ubiquitination and degradation via the proteasome. In the absence of Aux/IAA proteins, ARF proteins function as transcriptional activators or repressors, binding the AuxRE TGTCTC within the promoters of primary auxin response genes (27
). During normal plant development, the stability of Aux/IAA proteins is regulated by an auxin concentration gradient emanating from the shoot apex. Disrupting the function of Aux/IAA proteins or the genes controlling their stability results in numerous developmental abnormalities, including the loss of apical dominance, alterations in leaf development, and changes in floral promotion (41
On the basis of the present model for auxin signaling, we hypothesize that during a TMV infection, interaction with the viral replicase promotes the destabilization and/or inappropriate sequestration of PAP1, thus interfering with its function. Disruption of PAP1 function directed by the TMV replicase protein would occur independently of the plant's auxin gradient, resulting in the activation of ARFs and alterations in the transcription levels of specific auxin response genes. Consistent with this possibility, a significant portion (~30%) of the transcriptionally altered genes in TMV-infected leaf tissues contained multiple AuxREs within their promoter sequences (23
). Furthermore, experimental results indicate that TMV-altered AuxRE genes display auxin-induced expression trends similar to those observed in TMV-infected tissues (Table ). Microarray results also indicate that other genes containing AuxRE promoter sequences, including members of several primary auxin response gene families, such as SAUR (see SAUR-AC1 results in Table ), GH3, and other Aux/IAA proteins do not display transcriptional alteration in response to TMV (23
). Thus, the regulation of TMV-altered AuxRE genes (Table ) appears specific and not part of a genome-wide disruption in auxin sensing. Specificity in the effect of TMV on the auxin response system is further demonstrated by the inability of TMV to alter the localization of IAA10, a non-replicase-interacting Aux/IAA family member. Combined, these data support a link between TMV-altered AuxRE genes and the disruption of PAP1 stability or localization by TMV. Thus, TMV-altered AuxRE genes are candidates for additional studies directed at determining their role in the development of disease symptoms.
The induction of disease symptoms is likely to be complex, involving multiple interactions between host and pathogen components. PAP1 is only 1 of 29 predicted members of the Aux/IAA family of auxin-responsive transcription factors and shares between ~26 and ~67% sequence homology with the other members. Although IAA10, which has 41% homology with PAP1, did not interact with the TMV helicase, it is possible that other more closely related Aux/IAA members interact in a PAP1-like fashion. In addition, recent studies have determined that several auxin regulatory components, including ARF8, ARF10, and TIR1, are targets for micro-RNA (miRNA) regulation (6
). The ability of virus-encoded RNA-silencing suppressors to interfere with the miRNA-directed regulation of such components has also been correlated with the appearance of symptom-like developmental defects (7
). Thus, interaction of the TMV replicase with PAP1 likely represents only one avenue through which plant viruses can disrupt the auxin signaling pathway. The ability of TMV-V1087I to induce developmental symptoms, albeit reduced in severity, supports a role for other viral processes and interactions in the development of disease symptoms. At this time, we are investigating this possibility as well as the potential contributions of other Aux/IAA family members in the display of disease symptoms.
Interestingly, silencing PAP1 mRNA did not produce a detectable effect on virus replication or movement. Similarly, TMV-V1087I, a helicase mutant with reduced ability to interact with PAP1, replicated and spread at levels similar to those of the wild-type virus. Thus, the interaction between the TMV HEL domain and PAP1 is not rate limiting for virus function. This type of nonessential interaction may account for the differences between diseased and tolerant host responses. Both diseased and tolerant hosts show similar levels of susceptibility to a pathogen; however, only the diseased host displays significant damage (2
). In addition, disease and tolerant phenotypes in both host and pathogen backgrounds are heritable characteristics, suggesting the involvement of specific host-pathogen interactions. In fact, virus-induced disease severity often does not correlate with the ability of an infecting virus to replicate at high levels or spread rapidly within a specific host (29
). Therefore, nonessential interactions, such as the one between TMV replicase and PAP1, may play significant roles in determining disease severity.
Combined, these experiments suggest that the TMV replicase protein disrupts PAP1 function. One possibility is that this interaction destabilizes PAP1 through a ubiquitin-mediated process similar to the auxin-directed degradation of other Aux/IAA proteins. While virus-directed protein degradation has not been established as a disease mechanism in plants, it has been directly linked to disease development in several animal virus systems (4
). For example, human papillomavirus (HPV) E6 protein directs the degradation of the cellular tumor suppressor protein p53 as well as several membrane-associated guanylate kinases, contributing directly to the malignant progression of HPV-associated cancers (46
). Alternatively, the TMV replicase protein may simply sequester PAP1 protein and prevent its ability to localize to the nucleus. The precise mechanism by which TMV disrupts PAP1 function remains to be determined.