| Oomycetes owning unique taxonomic status,belongs to stramenopila.It is similar in morphology to fungi but evolved far from the fungus,it seriously threatening the global food production and the safety of natural ecosystems.Phytophthora is the most notorious of the oomycetes and is known as the "plant killer".For example,soybean root rot caused by Phytophthora sojae and potato late rot caused by P.infestans lead to billions of dollars in economic losses worldwide each year.At present,the control of Phytophthora disease mainly relies on the use of fungicides and disease-resistant varieties,but due to the rapid variability of Phytophthora,fungicide target genes and avirulence genes can produce various forms of variability to escape the virulence of pesticides and identification of new varieties.At the same time,in the process of interaction between plant and Phytophthora,for successful infection,Phytophthora will secrete a large number of effectors to play a toxic function and regulate plant immunity.Therefore,to master the mechanism of Phytophthora resistance and the mechanism of effector toxicity have a great significance to understand the phytosanitary mechanism in theory and the rational use of fungicides.In this study,the function of two genes of P.sojae in fungicide resistance and the toxic function of Phytophthora capsici effector RxLR207 were systematically analyzed,and the possible molecular mechanisms were explored.The main results and conclusions were as follows:The gene EGZ16653 of P.sojae may not be the target of metalaxyl but may affect mycelial growth after knockout.In the early stage of the study,six anti-metalaxyl strains were re-sequenced in the genomic level,and a possible anti-metalaxyl target gene EGZ16653 was obtained by bioinformatics analysis and PCR sequencing.After functional annotation,EGZ16653 was found as a glycosyltransferase.To further investigate whether EGZ16653 is the target of metalaxyl,we knocked out and overexpressed the gene in a wild-type strain and knocked it out in fungicide-resistant strain.After analyzing these transformants,it was found that these transformants obtained by knocking out in the wild-type and fungicide-resistant strains had a significant inhibitory effect on growth,while the overexpression transformants had no effection.After resistance test for overexpression transformants,it was found that the transformants were insensitive to metalaxyl compared with wild type,and it was hypothesized that EGZ16653 might not the target of metalaxyl.In order to investigate whether overexpression of EGZ16653 has an effect on other phenotypes,we observated mycelium morphology,tested pathogenicity and oxidative stress,and found that transformants did not differ in these phenotypes.P.sojae EGZ16653 may not be the target of metalaxyl but may affect mycelial growth after knockout.In the early stage of the study,six anti-metalaxyl strains were re-sequenced by genome,and a possible anti-metalaxyl target gene EGZ16653 was obtained by bioinformatics analysis and PCR sequencing.After functional annotation,EGZ16653 was found as a glycosyltransferase.To further investigate whether EGZ16653 is a target for metalaxyl,we knocked out and overexpressed the gene in a wild-type strain and knocked it out in fungicide-resistant strain.After analyzing these transformants,it was found that these transformants obtained by knocking out in the wild-type and fungicide-resistant strains had a significant inhibitory effect on growth,while the overexpression transformants had no effection.After resistance test for overexpressing transformants,it was found that the transformants were insensitive to metalaxyl compared with wild type,and it was hypothesized that EGZ16653 might not the target of metalaxyl.In order to investigate whether overexpression of EGZ16653 has an effect on other phenotypes,we observated mycelium morphology,tested pathogenicity and oxidative stress,and found that transformants did not differ in these respects.The P.capsici effector PcRxLR207 is required for its toxicity.Previous study have found that PcRXLR207 can cause cell death and can induce the accumulation of H2O2 to reduce the pathogenicity after inoculation of tobacco.To further evaluate the effect of RxLR207 on virulence of P.apsici,we knocked out RxLR207 using the CRISPR/Cas9 technique,and obtained two knockout transformants T11 and T14,T3 was a negative control.Compared with WT and T3,the morphology and growth rate of transformants T11 and T14 did not change.After inoculating Arabidopsis thaliana and pepper respectively,it was found that compared with the negative control and WT,the lesion of the knockout transformants was smaller and the pathogenicity decreased significantly,indicating that the effector PcRxLR207 is necessary for the toxicity of P.capsici.This conclusion lays a foundation for further study of the pathogenic mechanism of the effector,and provided clues for exploring the pathogenic mechanism of Phytophthora. |
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