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How to feed the world has become a huge challenge with the increasing world population. Potato, the most important non-cereal crop, is widely consumed as staple food in the Western world, and recently it has proposed to be also promoted as a staple food in developing countries like China. However, the global production of potato has greatly been hampered by a disastrous disease, called potato late blight. This disease is caused by the oomycete pathogen Phytophthora infestans, which triggered the great Irish famine in the 1840s. Besides chemical control, resistance (R) genes from wild relatives of potato have been introduced into modern cultivars by traditional breeding as well as by transgenic technology (GMO). However, most R genes that belong to the nucleotide-binding domain and leucine-rich repeat containing (NLR) class, which are generally rapidly defeated by the fast-evolving P. infestans population in the field. On the other hand, another form of disease resistance that constitutes the first layer of plant defense in the apoplastic space, has not received enough attention by breeders. This apoplastic defense is mediated by the plant surface immune receptors. These receptors can recognize microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) or apoplastic effectors of pathogens and trigger a defense response. MAMP/PAMP-triggered immunity (MTI/PTI) is believed to be more wide spectrum and more durable than the typical effector triggered immunity (ETI) that is mediated by cytoplasmic NLR receptors. In this thesis, I studied various apoplastic effectors from P. infestans and their receptors in the host, with the ultimate aim to achieve a broader and more durable resistance to late blight.
In chapter 1, I summarized the history of potato late blight resistance breeding, the current knowledge of the plant immune system, particularly the plant surface immune receptors and the effector biology in the age of genome sequencing.
To understand the surface immunity against P. infestans in potato, I first studied an ubiquitous but functionally unknown apoplastic effector family in Phytophthora- the PcF/SCR effectors. PcF (Phytophthora cactorum-Fragaria) was identified from P. cactorum, and the related effector SCR74 from Phytophthora infestans belongs to a highly diverse gene family. They represent small cysteine-rich (SCR) proteins which are normally up-regulated during infection. In Chapter 2, I collected all the annotated PcF/SCR proteins that share a PcF domain from public database. Sequence analysis, phylogenetic, genomic analysis as well as sub-cellular localization, mutagenesis, functional screening and disease tests were performed in this study. PcF genes are conserved in all tested isolates of P. cactorum and its orthologs from different Phytophthora species are sharing a co-linear genomic architecture. PcF can be recognized by a broad spectrum of different host plants, including strawberry, tomato and potato. In contrast, the SCR74 genes are exclusively present and expanded in P. infestans and under positive selection. They are secreted from haustoria and the cysteine residues are important for maintaining their function in the apoplast. Our effectoromics screening indicated that the SCR74 recognition is confined to wild potatoes. Collectively, this study provides a good example of the effectors from the same family possessing both MAMP/PAMP and effector features. This may lead to identification of multiple host surface immune receptors against these PcF/SCR effectors.
To accelerate the map-based cloning of plant surface immune receptors that perceive apoplastic effectors, such as SCR74 as described in Chapter 2, I developed a receptor-like protein (RLP) and receptor-like kinase (RLK) enrichment sequencing (RLP/Kseq) method (Chapter 3). Two diploid Solanum microdontum genotypes which respond to INF1 or SCR74-B3b, respectively, were crossed. The F1 population segregates for responses to INF1 and/or SCR74-B3b independently. I designed an enrichment bait library representing the RLP/RLK genes predicted from the potato reference genome S. tuberosum Group Phureja clone DM1-3 (DM). RLP/KSeq confirmed the localization of the INF1 receptor ELR on chromosome 12, and lead to quick mapping of the putative SCR74 receptor on chromosome 9. Our findings show that RLP/Kseq enabled rapid mapping of plant surface immune receptors and it is especially useful for crop plants with large and complex genomes.
To fine map the SCR74 receptor, in Chapter 4, I expanded the mapping population and developed more markers. The SCR74-B3b receptor was mapped to a 74kb interval based on the DM genome. The candidate genes include 3 G-type LecRK (G-LecRK) genes. To functional study the putative SCR74 receptor, homology-based cloning was firstly deployed to isolate candidate gene(s), from the SCR74-B3b responsiveness S. microdontum spp. gigantophyllum genotype GIG362-6. Later, I generated a bacterial artificial chromosome (BAC) library for GIG362-6. Three BAC clones covering the mapping interval were isolated and sequenced. I also used RNAseq data to provide expression data of these candidate genes. Attempts were made to perform complementation tests of these candidate G-LecRK genes by co-expression with the matching SCR74-b3b effector, however, I have not been able to confirm the identification of the SCR74-B3b receptor yet.
Chapter 5 is dedicated to the Pep-13/25, which are typical MAMPs/PAMPs from the transglutaminase GP42 of Phytophthora. The Pep-13/25 peptides are known to induce defense responses in parsley and potato, and were studied decades ago, but the receptor has remained undiscovered. Here, MAMP Pep-13/25 were functionally screened on wild Solanaceae species for cell death response. Various wild potatoes were found responsive to Pep-13/25, including GIG362-6 that was previously used for mapping of the SCR74-B3b receptor (Chapter 3 and 4). A population that is segregating for the response to Pep-25 was generated, and a bulked segregant RNAseq (BSA-RNAseq) method was successfully applied to locate the candidate Pep-25 receptor on chromosome 3 of the DM reference genome. Furthermore, I fine-mapped the Pep-25 receptor to a RLK locus. One BAC clone from the Pep-25 responding genotype GIG362-6 was isolated and sequenced. In addition, RNAseq was used for characterizing the gene expression level in the presence or absence of treatment with P. infestans. This work provides insight into the Pep-13/25 recognition in Solanaceae species and will most likely lead to the identification of a new MAMP receptor in potato.
In Chapter 6, I discuss the new findings of this thesis and put them in a broader picture. Four main topics are discussed: 1. What’s the boundary between MAMP and effectors, as well as between MPI and ETI? And do the surface immune receptors contribute to resistance breeding? I believe that the first layer of defense is definitely crucial for engineering more durable resistance to plant diseases, however more studies are needed to understand the whole picture of the plant immunity network; 2. Is the reproduction system of flowering plants evolve from the plant immune system? The latest knowledge of both systems was reviewed and compared, and based on current data, I suggest that SCR proteins and their receptors might be a “missing link” between the plant reproduction system and the immune system. 3. How to clone plant R genes in the era of genome sequencing? With the advancement of sequencing technologies, many genotyping-by-sequencing methods have emerged, as well as enrichment-based methods like RenSeq. I compare the pros and cons of each method and provide a roadmap to select the best methods to clone plant R genes.
Overall, this thesis provides: 1) new insights in the plant-microbe interaction in the extracellular space, 2) our newly developed RLP/KSeq methodology will accelerate the mapping and cloning of novel plant surface immune receptors, and 3) our findings will lead to identification of at least two novel surface immune receptors, which might contribute to more durable late blight resistance in potato.
|Qualification||Doctor of Philosophy|
|Award date||31 Oct 2018|
|Place of Publication||Wageningen|
|Publication status||Published - 2018|
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Lin, X. (Creator), Wageningen University & Research, 20 Sep 2018