Characterization of HRT Mediated Resistance to Turnip Crinkle Virus in Arabidopsis

The ability of a plant to resist an invading pathogen is sometimes regulated by a gene-forgene interaction between the products of a plant resistance (R) gene and a corresponding pathogen avirulence (avr) gene (39; 51). If either the plant or pathogen lacks the cognate gene, the plant's defense responses will be activated too slowly and/or weakly to prevent pathogen colonization. By contrast, when both Rand avr genes are present, a direct or indirect interaction between their products activates a signaling cascade( s) leading to disease resistance. Some of the defense responses induced in the inoculated leaf may include generation of reactive oxygen species (ROS), ion fluxes, protein phosphorylation /dephosphorylation, activation and/or synthesis of antimicrobial compounds (phytoalexins), accumulation of salicylic acid (SA) and the expression of pathogenesis-related (PR) genes (30; 35; 36;'41).A hypersensitive response (HR), in which necrotic lesions develop at the site(s) of pathogen entry, also frequently appears. Subsequent to these responses in the inoculated leaf, increased PR gene expression and SA content are often detected in the uninoculated portion of the plant. These increases correlate with the appearance of a systemic and long-lasting resistance to a widl:?variety of pathogens known as systemic acquired resistance (SAR; 30; 35; 36; 89; 116). Many studies have demonstrated that SA is a critical signal for the activation of disease resistance. SA-deficient plants, due to expression of the salicylate hydroxylase encoding nahG transgene, fail to develop SAR and exhibit increased disease susceptibility. The cpr, lsd, cim, ssi, snc and acd mutants of Arabidopsis, which contain elevated levels of SA, display enhanced disease resistance (35; 36; 41). By contrast, eds, sid and pad mutants, which display impaired SA synthesis, accumulation or transport (19; 77; 77a; 113), and nprl/niml mutants, which are insensitive to SA (12; 29; 42; 95) exhibit increased disease susceptibility. NPRI is a key transducer of the SA signal; however, an SA-dependent, NPRI-independent pathway(s) that regulates PR gene expression and/or resistance to certain bacterial or fungal pathogens also has been identified (10; 18; 20; 59; 77; 86; 88; 93; 117). Since SA treatment does not enhance disease resistance in npr1 mutants, it is presumed that a second, pathogen-induced signal works in conjunction with SA to activate the NPRI-independent pathway(s). R genes recognizing bacterial, fungal, viral, oomycete, nematode and insect pathogens have been identified and cloned (70; 102). Through this process, many R genes were shown to belong to tightly linked multigene families, and the loci for different R genes are frequently clustered on the chromosome (71). Within an R gene family, the various members usually specify resistance to different pathovars or biotypes of the same pathogen. However, different members of the HRT/RPP8 .andRx/Gpa2 gene families confer resistance to unrelated pathogens (25; 105). By comparing the sequences of different family members, it was concluded that unequal crossing over is an important mechanism for the generation of novel resistance specificities. Analysis of the predicted proteins encoded by all cloned R genes indicates that they can be divided into five categories (23). R proteins in the two largest categories contain a nuc1eotide.,binding site (NBS) fused to a leucine rich repeat (LRR) region. In prokaryotic and eukaryotic proteins, the NBS domain binds ATP or GTP and is important for catalytic activity. Nucleotide binding has not yet been reported for plant R proteins; however, they contain several conserved motifs associated with this activity. The LRR domain has been implicated in proteinprotein interactions and ligand binding in diverse proteins. Since this domain is highly divergent in R protein homologs, it may function as a key determinant of pathogen recognition. Some NBS-LRR proteins have a coiled coil (CC), frequently in the form of a leucine zipper (LZ), at their amino terminus (N-t). By contrast, other NBS-LRR proteins contain an N-t TlR domain which shares homology with the Drosophila Toll protein and the interleukin-I receptor (IL-IR) of mammals. Since IL-IR and the Toll protein are involved in activating inflammatory responses and innate immunity, the TlR region may be responsible for transducing a signal that induces plant disease resistance. . Pathogen recognition is likely the first step in the defense pathway; thus, R proteins are believed to function as direct or indirect receptors for the appropriate avr proteins. Using a yeast two-hybrid system, the tomato Pto and the rice Pi-ta proteins were shown to interact with their cognate avr proteins, avrPto and avr-Pita, respectively (47; 92; 99), and the Arabidopsis RPS2 protein formed an in vivo complex with avrRpt2 (58). However, other RJavr protein pairs have not yielded a detectable interaction (79). Thus, it was suggested that R proteins guard other plant proteins that are targets for avr proteins (27a; 102; 102a). Consistent with this possibility, the RPS2/avrRpt2 complex contains a third, unidentified protein of 75 kDa (58). In addition, Pto, which belongs to the serine/threonine protein kinase class of R proteins, requires Prf, which belongs to the CC-NBS-LRR R protein family, to activate defense responses (91). Moreover, RPMI, AvrRPMI and AvrB were all found to interact with a protein, termed RIN4, which positively regulates RPMI-mediated resistance and negatively regulates basal defenses against virulent pathogens (64a). Since RIN4 is phosphorylated in the presence of AvrRPMI or AvrB, it was proposed that RPMl "guards" the plant against pathogens that alter RIN4 activity, possibly through phosphorylation. Downstream of the recognition event, the signals activated by various Arabidopsis R proteins appear to converge into a small number of pathways (1). The pathway activated by rlRNBS- LRR proteins generally requires the EDSl (enhanced disease susceptibility) gene (81), while that activated by most CC-NBS-LRR proteins requires the NDRl (non-race specific disease resistance) gene (13). However, several CC-NBS-LRR R genes, including RPP8, RPPl3-Nd, and HRT, as well as RPP7 (which has yet to be cloned), signal resistance via a pathway(s) that is nearly or completely independent of EDSl and NDRl (7; 65; unpublished data). Evidence for multiple defense signaling pathways also comes from analyses of tobacco treated with salicylhydroxyamic acid (SHAM), an inhibitor of alternative oxidase and lipoxygenase (15; 16). A SHAM-insensitive pathway mediates SA-induced PR gene expression and resistance to a bacterial and a fungal pathogen, while a SHAM-sensitive pathway regulates SA-enhanced resistance to tobacco mosaic virus (TMV), cucumber mosaic virus (CMV) or potato virus X (PVX; 15; 16; 78). While significant advances concerning the resistance pathways activated by bacterial and oomycete pathogens have been made in recent years, our understanding of viral resistance is limited. To elucidate how plants resist viral infection, we therefore are studying the interactions between the model plant Arabidopsis thaliana, and turnip crinkle virus (rCV). rcv is a small icosahedral virus belonging to the carmovirus group. Its 4 kb genome, which consists of singlestranded, positive sense RNA, contains five open reading frames (ORPs). While most Arabidopsis ecotypes are susceptible to TCV infection, the Dijon (Di-O) ecotype is generally resistant (60; 96). From this ecotype, we isolated a rCV-resistant line (Di-I7) and a rcvsusceptible line (Di-3; 32). Following rcv infection, Di-I7 plants develop an HR on the inoculated leaves, accumulate SA and the phytoalexin camelexin, and exhibit increased PR gene expression in both local and systemic leaves (31; 32; 100). In comparison, the resistance associated with most other Arabidopsis/viral interactions does not involve these responses, but rather appears to be due to a lack of factors needed for viral replication or movement (14; 46; 54; 56; 57; 98; 111). Most (70 - 98%) of the TCV-infected Di-17 develop no further symptoms and the virus is localized to the lesions (32). The remaining plants develop disease symptoms on the uninoculated leaves that range from mild to severe. The variations in resistance levels appear to be due, at least in part, to the combined influences of environment and plant age atthe time of infection (32). In comparison to Di-17, Di-3, like susceptibl~ Arabidopsis ecotypes, fail to develop an HR following TCV infection. Di-3 also fail to accumulate camalexin, and both PR gene expression and SA accumulation are delayed and weak. Disease symptoms develop several days post inoculation (dpi) and increase in severity while the virus spreads throughout the plant in a defmed pattern (22; 32). By 21 dpi, Di-3 plants are dead. Genetic analysis indicated that HR development in Di-17 is regulated by a single dominant gene, termed HRT (HR to lCV; 31). Map-based cloning revealed that HRT encodes a CC-NBS.;LRR R protein with high homology to RPP8 and two RPP8 homologs (25). Expression of the HRT gene in TCV-susceptible Columbia (Col-O)restored HR formation after infection. However, most of these plants subsequently developed disease symptoms. Consistent with this finding, resistance to TCV infection was shoWnto be regulated by both HRT and a second gene, termed rrt (regulates resistance to lCV; 48). Since the rrt alleles found in Di-17 are recessive to those in Col-O,RRTlikely encodes a dominant, negative regulator (suppressor) of the TCV resistance pathway. Analysis of the HRT signaling pathway has revealed that HR formation and TCV resistance require SA, but not NPRI or the defense signals jasmonic acid (JA) or ethylene (48). We propose to i) clone and characterize RRTand ii)further dissect the HRT-mediated resistance pathway through both epistatic analyses with existing mutants and the isolation of new mutants. HRT will also be further characterized Hi)by domain-swapping type experiments to defme the regions required for interaction with the TCV coat protein (CP) and for initiating the SA-dependent defense signaling pathway(s) and iv)by using HRT-specific antibodies to determine its location and stability. The CP is the cognate avirulence factor recognized by HRr (25; 80; 87; 108; 121).