Functional Investigation Of Phytophthora Nitrogen Metabolic Pathways And Plant AGO4 During Interaction

In host-pathogen interactions, successful invasion by pathogens depends as much on their ability to overcome defensive mechanisms as their ability to utilize the available nutrient sources offered by plants, and the competition and utilization of nitrogen are one of the key factors between them. Meanwhile, host plants also have evolved complex immune systems to against microbial invasion, in which AGO proteins are the core component of the effector complex in sRNA-directed gene silencing, which is important for gene expression reprogramming process. A number of different aspects relating to host-pathogen interaction have been studied but little is known about the ways of Phytophthora utilizing nitrogen nutrition and the roles of AG04 in plant resistance. In this study, we have carried out preliminary studies of these two issues using P. sojae and the model plant A. thaliana as experimental materials, respectively.The main results and conclusions obtained are as follows:A genome-based identification and analysis of nitrogen metabolism genes in Phytophthora sojae revealed the evolutionary characteristics: Here, we identified the candidate genes for the uptake and assimilation of nitrogen as well as amino acid biosynthesis and degradation based on genome sequence of P. sojae, and compared identified candidates genes with other pathogenic oomycete and fungi. A total of 274 nitrogen metabolism related-genes have been identified from the genome database of P.sojae, and which was higher in number among all identified genes. Most of genes involved in nitrogen metabolism pathways have been identified in P. sojae, but the genes regulating the biosynthesis of lysine, phenylalanine and tyrosine were partially deleted. Exspecially,the gene expansion has occurred in GABA metabolic pathway, and the genes are up-regulated during the infection. Furthermore, multifunctional enzymes are extensively distributed in amino acid metabolism pathway of oomycete, which posses the different gene composition in some aspects compared with plant and fungi. These results showed that the nitrogen metabolism of oomycetes is similar to other species, but there are some differences in the number distribution and gene structures between them. P. sojae could acquire and utilize different nitrogen sources to synthesize the most of amino acids beside lysine,tyrosine and phenylalanine. Moreover, the production of disease-induced enhanced plant resistance responses GABA and uric acid could serve as the nutrient sources to P. sojae,,leading to the concept that successful pathogens may hijack potential plant defences for their nutritional benefit during colonization.Nitrogen acquisition is involved in pathogenicity of P. sojae during infection: In this study, we analysis of the available genome sequences of P. sojae reveal the presence of 8 ammonium transporters,16 nitrate/peptide transporters and 78 amino acid transporters respectively, and which are significantly higher than the other tested oomycete and fungi,indicating that transporter genes of P. sojae undergo the obviously expansion. However, the oomycete-specific ammonium transporters are identified and divided into Rh group. In contrast, oligopeptide transporters in plant and fungi are in general, with no homologous genes in oomycete. Meanwhile, most of transporters are up-regulated in the infection process which have been confirmed by qRT-PCR. Analysis of P. sojae growth on N-limiting artificial medium displayed that it can be grown on the medium with different nitrogen sources and there were some differences among them, such as cysteine significantly inhibited the growth of P. sojae. The content of free amino acids has increased in infected leaves by P. sojae, and the most significant of which is glutamate. Moreover, we find that ammonium transporter PsRh1 is located on the plasma membrane and induced by inorganic nitrogen. Subsequently, PsRh1 and a cationic amino acid transporter PsCAT3 for functional characterization by homology-dependent gene silencing. The silencing of two genes in P. sojae lead to decline of pathogenicity, revealing that two genes are important for pathogenesis on host soybean cultivars. These results suggest that P. sojae could utilize various nitrogen sources,which may somehow manipulate plant metabolism to maintain and even increase the apoplastic concentration of nitrogen compounds, while nitrogen acquisition of P. sojae may play a important role during infection.PsAAT3, an oomycete-specific aspartate aminotransferase, is required for full pathogenicity of the oomycete pathogen P. sojae: In this study, we bioinformatically identified P. sojae aspartate aminotransferases (AATs), which are key enzymes that coordinate carbon and nitrogen metabolism. We demonstrate that P. sojae encodes much more AATs than the tested fungi. Some of the AATs contained additional prephenate dehydratase and/or prephenate dehydrogenase domains in their N-termini, which are unique to oomycetes. Silencing of PsAAT3, an infection-inducible expression gene, reduced P.sojae pathogenicity on soybean plants and affected the growth under N-starving condition,suggesting that PsAAT3 is involved in pathogen pathogenicity and nitrogen utilisation during infection. Our results suggest that P. sojae and other oomycete pathogens have distinct amino acid metabolism pathways and that PsAAT3 is important for its full pathogenicity.ARGONAUTE4 is involved in Arabidopsis resistance to P. capsici and Verticillium dahlitae with different Pathway: In this study, we report that A. thaliana ago4-2 mutants showed enhanced disease resistance towards the oomycete pathogen P. capsici, whereas the defenses that protect against fungi V. dahliae are reduced. Meanwhile, the ROS accumulation and callose deposition confirmed the results. In addition, AG04 is downregulated by P. capsici infection, but highly induced by infection with V. dahliae,implying a complex role of AG04 in plant immune responses. Interestingly, we show that loss of function in other components of the pathway operating upstream of AG04, such as NRPE1, RDR2 and DCL3, or operating downstream, such as DRD1 and NRPD1, enhance resistance to P. capsici. Nevertheless, all the mutants does not compromise resistance to the fungul pathogen V. dahliae. Taken as a whole, our data illustrate that AG04 may works independently of other components of the RdDM pathway in mediating resistance to V dahliae. In contrast, the RdDM pathway is required for AG04 in the negative regulation of Arabidopsis defense against P. capsici.