Resistance to Potato Virus X in Potato Conferred by Rx1 and Rx2

The Rx loci in potato, Rx1 on chromosome V and Rx2 on chromosome XII, confer resistance to PVX in the absence of necrotic cell death. Rx-mediated resistance results in a very rapid arrest of PVX accumulation in the initially infected cell. In contrast to HR-associated resistance, Rx-mediated resistance is active in protoplasts. When protoplasts isolated from resistant (Rx) and susceptible (rx) potato genotypes were inoculated with PVX and TMV, Rx protoplasts showed<100-fold less PVX RNA accumulation, relative to a positive control using TMV. When TMV was coinoculated with PVX, TMV RNA accumulation was also reduced to a level comparable to PVX in resistant protoplasts, demonstrating that once induced, the resistant response can target viruses other than the elicitor virus. Rx1, isolated from tetraploid potato by map-based cloning, encodes a 107.5-kD CC-NBS-LRR protein. Rx1 and Rx2 show the same specificity for the PVX CP, extremely similar nucleotide sequence, and similar linkage with resistance to Globodera. Transgenic experiments demonstrated that the response to PVX in Rx-containing genotypes can be altered depending on the mode of expression of the viral CP.

Transgenic potato or tobacco plants expressing Rx show extreme resistance against PVX. When the PVX CP is constitutively expressed in the same plants, HR is observed, indicating that the amount of CP in the plant cell determines the macroscopic host response. Constitutive gain-of-function Rx mutants in which cell death is activated in the absence of viral CP were obtained by random mutagenesis. Sequence analysis revealed that most of the constitutive gain-of-function mutations occurred in or near the conserved NBS-LRR sequence motifs. It is not clear whether this phenotype is resulted from release of negative regulation by the LRR and adjacent sequences or introduction of an incompatibility between the domains such that they are no longer held inactive. In experiments designed to determine the biochemistry of Rx function, segments of the protein were expressed independently in an elegant system where phenotypic response could be easily assayed. PVX CP-dependent HR was observed after fragments of Rx (CC and NBS-LRR domains) and PVXCP were expressed transiently in N. benthamiana via agroinfiltration.

These results indicate that a functional Rx protein can be reconstituted through physical interactions between domains, even when the domains are expressed in different molecules. Furthermore, PVX CP disrupted the interaction between these Rx derived domains. The current model suggests that CP recognition induces sequential conformational changes in Rx, disrupting intramolecular interactions, thereby activating Rx-mediated signaling. Experiments using virus-induced gene silencing (VIGS) showed that Rx-mediated resistance does not require EDS1 and RAR1. Bieri et al., however, have shown that silencing Rar1 actually reduces the levels of Rx. Therefore, Rar1 is likely a cochaperone required to varying degrees by different R proteins. Silencing of tobacco MAP kinase kinase kinase (MAPKKK) interferes with the function of the Rx gene. Similar to results described above for the N gene, silencing SGT1 also compromised Rx-mediated resistance, and HSP90 is required, presumably acting as a cochaperone to stabilize Rx.