| Potato (Solarium tuberosum L.) belongs to the Solanum section Petota that originates from the Andes in South America. The cultivated potato is the fourth food crop in the world after maize, wheat and rice. Potato suffers from one of the most destructive diseases late blight which is caused by oomycete pathogen Phytophthora infestans (Mont.) de Bary. Until today, P. infestans still costs about $6.7 billions of yield loss, seriously threatening global food security. The most sustainable strategy to protect potato from late blight is to breed broad spectrum resistant cultivars. However conventional potato breeding is slow. Moreover, P. infestans has a remarkable capacity to rapidly adapt to the resistance in host plants which makes the introduced resistance genes quickly defeated. Therefore, potato breeders urgently need to adopt new strategies and techniques for durable resistant varieties against late blight. Effectoromics, defined as a high-throughput, functional genomics approach that uses effectors for probing plant germplasm to detect R genes, has proven to be a potent contributor to modern breeding. Now, the strategy has been successfully pioneered for P. infestans and potato, and provided a theoretical basis for durable resistance breeding of potato. In this study, we made a selection of effector candidates and subjected them to a large functional screening on various potato resources. We performed in depth functional analysis of promising avirulence candidates, such as effectors Pi23008, Pi22724 and Pi22798. The main achievements are shown as below.1. Screening of P. infestans avirulence gene candidatesThe 68 up-regulated RxLR genes from P. infestans effectors were used as effector candidates. We used the 68 RXLR effectors to screen 116 potato genotypes by agroinfection. As a result, ten RxLR effectors were identified as AVR candidate genes based on the hypersensitive cell death response. They are Pi23008 (a AVR2 family number), RD39 (a AVR-blbl family number), RD2 (a cell death inducer), and other effector, such as Pexl47-2, RD8, RD49, P110232, Pi11484, Pi07555 and Pi22724. In the meantime, we also tested the 68 RxLR effectors in N. benthamiana by agroinfiltration. Interestingly, we found one effector Pi22798 that can induce cell death in Nicotiana species.2. Mining of Rpi-R2 homologs and its recognition to AVR2 family membersIn the identified ten AVR candidate genes, we found that Pi23008 belongs to the AVR2 gene family. Actually, in the P. infestans genome, there are 14 AVR2 family members in total that can be classified into three groups. And we selected six family members of them together with Pi23008 for further study. To characterize their function, we also cloned four Rpi-R2 homologs from four diverse potato genotypes. We made a matrix of the seven AVR2 family members and four Rpi-R2 homologs, and tested each combination by agro-co-infiltration in N. benthamiana. As a result, we found that three combinations could induce HR responses, and only Pi23008 and Pi 13940 could be recognized. In addition, we proved that that only recognized AVR2 family members contain a nuclear export signal (NES).3. Structural, functional and recognition analysis of Pi23008 and Pi 13940To study the essential domains for the recognition of the identified AVR2 family member Pi23008, we cloned its homologs from nine P. infestans isolates. Pi23008 homologs show polymorphic changes in the 95th and 98th aa. By point mutation analysis, we confirmed that the two amino acids play a great role in Pi23008 induced HR. In addition, we also identified another key amino acid (109th aa) for the recognition of Pi13940, another A VR2 family member.Previous report shows that StBSL1 is required for Rpi-R2-mediated perception of AVR2 and resistance to P. infestans. AVR2 associates with BSL1 and mediates the interaction of BSL1 with R2 in planta, through the formation of a ternary complex. To see if BSL1 is also the target of the two recognized AVR2 homologs (Pi23008 and Pi13940), we tested the interaction of StBSL1 with each of them by yeast two-hybrid. We found that only Pi13940 but not Pi23008 can interact with StBSL1. The interaction between Pi 13940 and StBSL1 was also reconfirmed by BiFC assay. VIGS of BSL1 specifically perturbed Pi13940 triggered HR, but not Pi23008 triggered HR. Our results suggest that StBSLl is required for Pi13940 induced HR, wihle Pi23008 may have another potent target to induce the recognition by Rpi-hjtl.1.4. Functional analysis of effector Pi22798In the functional screening of P. infestans effectors, we also identified a potential avirulence gene Pi22798 that can trigger cell death in both N. benthamiana and N. tabacum. Strikingly, we found that transient expression of Pi22798 in N. benthamiana can also enhance P. infestans infection. And Pi22798 is up-regulated during the early infection about 24 hours after the inoclution of P. infestans in potato. Pi22798 homologs from 11 diverse P. infestans isolates are structurally and functionally conserved, all of which can induce cell death in N. benthamiana except that the homolog from Ljx18 induces a delayed cell death. These results imply that Pi22798 could be a contributor to the virulence of P. infestans. We further proved that P122798 triggered cell death relies on its localization to the plant nucleus and is dependent on the SGT1-mediated signal pathway. Moreover, Pi22798 triggered cell death can be suppressed by P. infestans effector AVR3b but not AVR3a or any other tested effectors. In all, our work suggests that the potential avirulence gene Pi22798 is also a contributor to the virulence of P. infestans and how it could be involved in modulating plant immune system. |
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