| Potato(Solanum tuberosum L.), which originates from the Andes in South America, is the fourth most important food crop in the world. Potato plays an important role for both human consumption and industrial purpose mainly for starch production. However, it suffers from various diseases, which cause huge economic losses every year. One of the most devastating diseases is late blight caused by the Irish potato famine agent Phytophthora infestans. During past years, many resistance (R) genes have been cloned and some of them have been introgressed into several potato cultivars by sexual crossing. Yet it was found that the potato with an introgressed single R gene normally could not last for long. Sometimes, the new cultivar was defeated by the fast evolving P. infestans even without going into the commercial pipelines. So we can not only rely on a single R gene and it is urgent to exploit more effective ways to provide durable resistance.Although most cloned R genes have been defeated by P. infestans, the virulent isolates are not everywhere. Proper use of known R genes and rapid cloning of new Rpi-genes could still make contributions to resistance breeding based on their stronger resistance. The traditional resistance breeding is slow and inefficient, which is hard to follow the evolution speed of P. infestans. Encouragingly, effctoromics has recently been proved a successful strategy to understand and achieve resistance to late blight in agriculture. In Chapter2, we provided suggestions in the application of effectoromics, in the form of "Do’s and Don’ts". Briefly, we summarized seven advantages in the Do’s and three practical notes in the Don’ts.Agroinfiltration and PVX agroinfection are two efficient transient expression assays routinely used for effctoromics. In Chapter3, we presented our protocols of the two assays in both potato and Nicotiana benthamiana. The protocols themselves are not complicated, but one may need more experience to analyze the results. We shared our long-term experience for scoring and also discussed about the advantages and disadvantages of both assays in effectoromics application.Defense-responsive genes are involved in the downstream signaling pathway and not directly interacting with pathogens. Thus they do not put too much selection pressure to P. infestans and are very likely to be more durable. To identify the key defense-responsive genes to potato late blight, we performed a functional screening of63selected candidate genes by VIGS (virus-induced gene silencing) on both Nicotiana benthamiana and potato (Chapter4). This treatment was followed by detached leaf assay and assessment of the resistance level. Our results led to the identification of two genes, a lipoxygenase (LOX; EC1.13.11.12) and a suberization-associated anionic peroxidase, which may play a role in quantitative resistance to potato late blight. Plants rely on two layers of immunity to defend against pathogens. Despite the defeated intracellular R genes, the other layer of immunity occurs at the cell surface. Since surface receptors can recognize conserved pathogen associated molecular patterns (PAMPs), they are considered to be more durable. For the potato-P. infestans pathosystem, this layer of immunity has not yet been explored, mainly because the quantitative resistance phenotype conferred by PAMP-triggered immunity is hampering map-based cloning approaches of surface receptors. Taking advantages of effectoromics assays, clear phenotypes could be obtained for recognition of elicitins, a family of proteins of P. infestans that are recognized as oomycete PAMPs. Recently, a receptor-like protein ELR (elicitin response) was successfully cloned from the wild potato species Solanum microdontum. Based on ELR, we studied the first layer of immunity in potato. In Chapter5, we performed more than three repeats of disease tests on ELR transgenic potato cultivar Desiree. Promising results showed that overexpression of ELR could enhance the potato resistance to late blight. Furthermore, we also found that Desiree::ELR could recognize a broad spectrum of Phytophthora elicitins and induce defense responses.In Chapter6, we further analyzed the natural variation of ELR in different plant species. On the map in SolRgene website, we found that INF1-responding wild potato species are distributed in both Central and South America. From those species, we successfully cloned7ELR orthologs, which show high levels of amino acid sequence identity with ELR. By testing these ELR orthologs with various elicitins, we found that patterns of elicitin-triggered defense are conserved in both Solanum and Nicotiana species. Moreover, we proved that INF1-triggered hypersensitive response (HR) can also be suppressed by AVR3aKI in a wild potato species(Solanum hjertingii349-3), which has been shown in N. benthamiana before. All these data indicate that the elicitin signaling pathway is conserved in Solanum and Nicotiana. This finding would accelerate further study of elicitin signaling pathway and application of apoplastic immunity in potato and other crops.In all, our study has led to1) useful suggestions for applying effectoromics,2) identification of several potential useful defense-responsive genes,3) proving that the newly cloned INF1receptor ELR can enhance resistance to P. infestans and induce broad-spectrum defense responses to different oomycete elicitins, and4) discovering that the elicitin signaling pathway is conserved in Nicotiana and Solanum species. The knowledge we obtained may be valuable for potato resistance breeding. |
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