Molecular Pathogens, 2025, Vol.16, No.5, 207-216 http://microbescipublisher.com/index.php/mp 210 3.3 RNA silencing and small RNA regulation RNA silencing (RNAi) is one of the unique and important innate immune mechanisms in plant antivirus. Plant cells are able to recognize and eliminate exogenous viral RNA by producing small interfering RNA (siRNA), thereby achieving the effect of "RNA against RNA". After the virus invades, the double-stranded RNA (dsRNA) formed by its genome or replication intermediates will be recognized and cleaved into small fragments of 21-24 nt siRNA by the plant's Dicer-like endonuclease (Andika et al., 2015). Subsequently, these virus-derived siRNA (vsiRNA) are loaded into the RNA-induced silencing complex (RISC), directing the complex to perform sequence-specific cleavage or translational inhibition of viral RNA, thereby preventing viral proliferation. In potato materials that are resistant to PVY, high levels of accumulated PVY-specific siRNA are often detected, and their presence is closely related to reduced virus titers. RNA silencing is a universal resistance pathway against almost all RNA viruses and some DNA viruses, functioning at all stages of viral infection (Choudhary, 2021). 4 Identification and Functional Analysis of Potato Virus Resistance Genes 4.1 Mining and classification of resistance gene resources Potato's antiviral genetic resources mainly come from its wild relatives and traditional varieties. In the long process of evolution, some wild species have gradually accumulated specific resistance alleles to resist viral infections in nature. Through distant hybridization, these genes have been introduced into cultivated species, providing valuable materials for breeding. According to their mode of action, antiviral genes can be roughly divided into genes that confer high resistance (immunity), usually dominant NLR genes; genes that confer moderate resistance or disease tolerance, which may be secondary effect genes or quantitative trait loci (QTL) (Meade et al., 2020); and regulatory genes related to broad-spectrum resistance, etc. Using molecular marker-assisted selection (MAS), researchers screened and classified the resistance genes of numerous potato germplasm, and conducted PCR testing on 103 potato varieties (lines) at home and abroad. The results found that the combinations of resistance genes carried in different materials were significantly different. Some varieties contain multiple antiviral genes at the same time, showing compound resistance (Gadjiyev et al., 2020). 4.2 Progress in research on key resistance genes Among numerous antiviral genes, Rx and Ry series genes have been intensively studied due to their remarkable resistance phenotypes and wide breeding applications. The Rx1 gene is one of the earliest cloned potato antiviral NLR genes, conferring extreme resistance to PVX. As early as the 1990s, researchers isolated the Rx1 and Rx2 genes from (S. tuberosum) ssp. andigena through map-based cloning. The proteins they encode contain the typical CC-NB-LRR structure. Rx1 recognizes the PVX coat protein CP and triggers a rapid resistance response, preventing PVX from spreading in the host. Follow-up studies found that the efficiency of Rx1-mediated resistance is regulated by the intracellular RanGAP2 protein: RanGAP2 can interact with the CC domain of Rx1 (Sukarta et al., 2021) and is a cofactor necessary for Rx1 function. 4.3 Case study: research on the molecular mechanism of PVX resistance mediated by Rx1 gene In order to understand the role of resistance genes more intuitively, the Rx1-PVX interaction is used as an example for analysis. When potatoes carry the Rx1 gene, they show extreme resistance to PVX. Even if they are inoculated with high-concentration virus juice, no symptoms will appear. The virus is restricted in the plant near the contact site, and no systemic infection occurs (Figure 2) (Richard et al., 2021). At the molecular level, Rx1 is activated early in PVX invasion, and its LRR region is believed to directly or indirectly detect the coat protein (CP) of PVX. Activated Rx1 rapidly oligomerizes and recruits costimulatory factors such as RanGAP2 to form a resistance complex. Subsequently, a local HR response is generated in cells surrounding the infection site, and a very small number of infected cells undergo programmed death, "uprooting" the virus (Slootweg et al., 2010). However, unlike many HR-type disease resistances, Rx1-mediated HR is very localized and mild, and no necrotic spots can be seen on the plant macroscopically. This may be due to the fact that the downstream signal intensity of Rx1 is strictly regulated, making it sufficient to defend against viruses but not injuring innocent people.
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