| Oomycetes are fungal-like eukaryotic organisms that are classified into the kingdom of Stramenopila.Oomycetes are widely considered as major threats for agriculture and natural ecosystems.Phylogenetic studies have shown that oomycetes are distantly related to true fungi.Among oomycetes,the genus Phytophthora(which means 'plant killer' in Greek)harbors some of the most destructive plant pathogens of dicotyledonous plants.For example,Phytophthora sojae is an oomycete and a soil-borne soybean pathogen that causes 1-2 billion dollars in damages globally every year.Late blight caused by Phytophthora infestans is the most well-known plant disease epidemics that led to the Irish potato famine in the nineteenth century.Effective managements of the disease includes host resistance and appropriate usage of fungicides.However,owing to the rapid evolution,its avirulent genes and fungicides targeted genes evolved many strategies to escape host recognition and toxicity of fungicides,resulting in the loss of host resistance and fungicide resistance.Therefore,it is of great significance to accurately undersdand population structure,pathogenicity,fungicide resistance and genetic variability of Phytophthora for breeding plant broad-spectrum resistance cultivars and application of fungicides.In this study,we selected a diverse pathotype population and fungicide resistant population of P.sojae strains for genome re-sequencing,systematically revealed the variation mechanism of avirulence genes,identified the target genes related to fungicide resistance.In addition,we have explored two important virulence-related genes.The main contents are as follows:Genome resesquencing of a diverse pathotype population revealed the genetic basis of virulence adaptation in P.sojae.Genome resequencing and analysis were performed using 25 different pathogenic strains of P.sojae strains mainly from China and the United States,.First,population structure analysis reveals two distinct P.sojae lineages of G1 and G2,and G2 only fcontains isolates from China.Second,whole genome single nucleotide polymorphism(SNP)analysis revealed that there were many SNPs in the promoter and coding region of the gene,accounting for 60.41%of the total SNPs.Non-synonymous mutations in the coding region accounted for 61.94%of the total mutations in the coding region,of which 1.72%leading to the early termination of encoded protein and 0.48%leading to the extension of encoded protein sequence,indicating that the point mutation is an important driving force for the evolution of P.sojae genome.Third,RxLR(R:Arginine;x:Any amino acid;L:Leucine)effectors play an important role in the pathogenesis of Phytophthora,and 374 RXLR effectors have been identified in P.sojae previously.Here,we identified 471 RxLR effectors,among which only 131 RxLR effectors are conserved.We found that RxLR effectors have a faster evolution rate and highly varied among strains,suggesting that RxLR effector variations contribute to the virulence adaptation of P.sojae.Fourth,except for known variants,we found several novel variants in avr genes,including gene mutations,gene deletions,gene silencing,pseudogeneization,and gene copy number variation,indicating that P.sojae posses several means to escape host immunity.Last,five plant-to-P sojae race-specific horizontal gene transfer(HGT)events were identified.For example,the glyceraldehyde 3-phosphate dehydrogenase gene of PS6 strain,the oligosaccharyl transferase gene of PS5 strain,the mitochondrial uncoupling protein 5-like gene of PS13 strain,and the unknown functional genes of PS2 and PS28 strains,declaring isolate-specific HGT contributes to environment adaptation in P.saoje.These findings present several means adapted by P.sojae for virulence and provides a valuable resource for future studies in resistance breeding and fungicides application.Genome resequencing of a diverse fungicide resistant population revealed the genetic basis of fungicide adaptation in P.sojae.Fluopicolide and metalaxyl are common fungicides that have excellent effects on oomycetes.However,the genetic basis of resistance to these two fungicides is not yet known.To investigate the genetic basis to fluopicolide and metalaxyl,we obtained the strains of P.sojae with different resistant levels to fluopicod and metalaxyl by the fungicide acclimation methodand used 15 strains were subjected to genome resequencing.The potential genes related to fungicide resistant were identified using the non-synonymous SNPs associated with gene coding region.The results showed that the mutations of Visinin protein V172M,Iron-sulfur cluster assembly I50V,keratin associated protein W25S or E28L/V and Tripartite motif containing protein L73P may mediate the resistance of P.sojae to fluopicodate.However,Alpha-1,3-glucosyltransferase V272L mediate the resistance of P.sojae to metalaxyl.The above results provide possible novel target genes for the study of the fungicide resistance of P.sojae and will lay a theoretical foundation for the evaluation of Phytophthora resistance and the rational use of fungicides.Autophagy plays an important role in the development and pathogenesis of P.sojae.Autophagy is an evolutionarily conserved mechanism in eukaryotes with roles in development and the virulence of plant fungal pathogens.However,few reports on autophagy in oomycete species have been published.Here,we identified 26 autophagy-related genes(ATGs)belonging to 20 different groups in P.sojae using a genome-wide survey,and core ATGs in oomycetes were used to construct a preliminary autophagy pathway model.Autophagy in P.sojae was detected by electron microscopy that more autophagosomes accumulated in the cytoplasm in rapamycin treatment hyphal cells during rapamycin treatment.Expression profile analysis revealed that these ATGs are broadly expressed and that the majority of them significantly increase during infection stages,suggesting a central role for autophagy in virulence.These results suggested that autophagy might play essential roles in both the development and infection mechanism of P.sojae.Filamentous pathogens encode a class of conserved unconventional effectors that control plant immunity.Salicylic acid is an important plant defense hormone and plays a vital role in plant immunity.Plant pathogen can interfere with the accumulation of salicylic acid in plants by secreting effectors,thereby controlling plant immunity.However,few mechanisms have been reported to interfere with plant salicylic acid accumulation.In this paper,the isochorismatase homologous protein Ps108159 was identified in P.sojae.After functional annotation,signal peptide prediction,conserved catalytic sites and secondary structure analysis,we speculated that Ps108159 may be an unconventional secretion effector.It synthesizes the precursor of isochorismate by targeting plant salicylic acid and interferes with the accumulation of salicylic acid in the plant,thus controlling the plant immunity and achieving the purpose of infection.Therefore,it was named as PsIscl.Further studies revealed that there was no sequence polymorphism of PsIscl in 29 P.sojae strains,suggesting that Pslscl may be a necessary virulence factor for pathogenicity.In addition,we identified a single copy of the isochorismatase homologue protein in most of the plant filamentous pathogens,indicating that isochorismatase interferes with plant salicylic acid accumulation as a general strategy for plant filamentous pathogens to manipulate plant immunity.These findings continue to reveal new pathogenic mechanisms of filamentous pathogens,and also provide new ideas for the identification of unconventional effectors and the control of filamentous pathogens. |
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