Function Of RxLR23 And Key Developmental Regulatory Gene FtsZ From Phytophthora Capsici

Phytophthora capsici is an important plant pathogen.In addition to infecting peppers,it can infect Solanaceae,Cucurbitaceae,and Leguminous plants such as tomato,eggplant,watermelon,and pumpkin.It has evolved far from fungi and belongs to the Stramenopila.It is difficult to prevent and control P.capsici using common fungicides.Therefore,it is easy to cause a variety of vegetable disease outbreaks and epidemics,and causes each year serious harm and loss to China and even the global vegetable industry.P.capsici can secrete a large number of effectors during the infection process,and transfer the effectors into plant cells through the aspirator,thereby suppressing the host's basic immunity.Oomycete cytoplasmic effectors mainly include RxLR and CRN.At present,little information is accumulated on the recognition of plant proteins by these effectors,and the research on how the effectors target proteins and damage the plant immune system is relatively weak.In addition,P.capsici sporangia and zoospores are important infectious structures,however,there is less accumulated data on functional genes regulating related key infectious structures and their regulatory mechanisms.?1?RxLR23 induced host immunity and PcFtsZ2 regulation of infectious development and pathogenic function have been thoroughly studied through molecular genetics technology,and outstanding innovative results have been achieved,with details as follows:It was clarified that RxLR23 regulates the pathogenicity of early infection of P.capsici.It was confirmed that the expression level of RxLR23 is not directly related to the four key developmental stages of P.capsici?mycelium,zoospores,cysts,and germinating cysts?,but it is highly expressed at 1.5 hours after the infection of the P.capsici using qRT-PCR,so RxLR23 should be a key gene for pathogenic activity in early stage of infection.It was proved that RxLR23 effectively regulated the pathogenic function of P.capsici.On this basis,CRISPR/cas9 was used to knock out RxLR23 to obtain multiple mutants.By measuring the pathogenicity colony development of the mutants,it was found that RxLR23 does not regulate the growth and development of P.capsici,and significantly affects the pathogenicity of the pathogen.It is further proved that the early upregulation of RxLR23 significantly regulates the pathogenicity of P.capsici.It was revealed that RxLR23 mainly performs HR toxic function in the nucleus.K⁹³M³²⁰ act as key amino acids for HR caused by RxLR23.RxLR23,_NLSRxLR23,_NESRxLR23 were inoculated on Nicotiana benthamiana using gene mapping and inoculation technology.As a result,RxLR23 was distributed in the cytoplasm and nucleus._NESRxLR23caused HR to be significantly weaker than RxLR23 and _NLSRxLR23.Meanwhile,HR mainly occurs in the nucleus,indicating that HR caused by RxLR23 is mainly concentrated in the nucleus.The sequence differences of RxLR23 in the strong pathogenic strain SD33,the medium pathogenic strain YN07 and the weak pathogenic strain Aug0202 were compared and analyzed using gene cloning and sequencing technology.Compared with the SD33 RxLR23sequence,it was found that the K⁹³M³²⁰ were mutated of medium and weak pathogenic strains,namely SD33(RxLR23K⁹³M³²⁰),YN07(RxLR23R⁹³M³²⁰)and Aug0202(RxLR23R⁹³R³²⁰).RxLR23K⁹³M³²⁰ was mutated into RxLR23R⁹³R³²⁰,RxLR23R⁹³M³²⁰,and RxLR23K⁹³RM³²⁰R respectively,and then N.benthamiana were inoculated with these RxLR23K⁹³M³2020 and other five RxLR23 alleles.As a result,the HR degree and nucleolar localization intensity of the three mutants were not significantly different from those of RxLR23 of YN07 and Aug0202,which caused HR to be weaker than RxLR23K⁹³M³²⁰.It was proved that K⁹³M³²⁰ were key functional amino acids that regulates HR response caused by RxLR23.Based on RxLR23 targeting CaERD15La,it reveal the mechanism by which RxLR23 induces the host plant immunity.Early response dehydrated 15 like proteins?CaERD15La?were screened for the RxLR23 interaction protein through Y2H,Co-IP,and BiFC.RxLR23 do not interact with CaERD15La homologous proteins CaERD15Lb and CaERD15Lc,and PABP which was interact with ERD15L.Furthermore,it was further proved that two effectors RxLR19781 and RxLR121504 which have same function as RxLR23 do not interact with CaERD15La.This proved that RxLR23 specifically interact with CaERD15La,and the co-localization of RxLR23 and CaERD15L further proved the interaction between them.It was further proved that RxLR23 can effectively inhibit the infection of P.capsici.It was found that RxLR23 could effectively inhibit zoospore infection of P.capsici,and its inhibitory effect is significantly higher than the other five mutant genes.In order to prove that this inhibition is related to the interaction of CaERD15La,DAB staining revealed that the interaction of RxLR23K⁹³M³²⁰ and CaERD15La can accumulate and release more ROS.Therefore,we conclude that the interaction of RxLR23K⁹³M³2020 and CaERD15La can release more ROS and promote the inhibition of P.capsici infection.ERD15La is a positive regulator of immunity.We carried out this experiment by silenced or overexpression NbERD15La of N.benthamiana.To further prove this conclusion,We silenced and overexpression NbERD15La,and found that silenced NbERD15La can reduce the HR response induced by RxLR23^K93M320,and overexpression of NbERD15La can reduce the infection of P.capsici.Meanwhile,N.benthamiana were inoculated with zoospore and culture filtrate of P.capsici,WRKY7,WRKY8,and NbERD15La could upregulation during infection,suggesting that NbERD15La acts as a positive regulator of immunity.RxLR23 activates a plant defense pathway that is centrally regulated by ERD15La.Because of ERD15L gene functions as a common regulator of ABA and SA response pathways,to further validate whether the expression of RxLR23 activates SA plant defense pathway via its interaction with ERD15La.We treated the silenced and wild type N.benthamiana with SA,then inoculated with RxLR23,and found that the ability of inhibitation to P.capsici was enhanced with SA treatment,and SA treatment of wild type had a stronger inhibitory ability.At the same time,inoculation with RxLR23 can further increase the ability of N.benthamiana to inhibit P.capsici.Comprehensive analysis of test data strongly proves that the interaction of RxLR23 and ERD15La plays an important role in plant defense responses,and that RxLR23 activates the SA defense pathway regulated by ERD15La and participates in plant defense activities and resistance processes.?2?The prokaryotic cytoskeletal protein FtsZ plays a central role in bacterial cell division.In rod-shaped bacteria such as Bacillus subtilis and Escherichia coli,FtsZ assembles into a ring shaped structure?Z ring?at midcell that establishes the location of the future division site and serves as a platform for assembly of the division machinery.As cell division progresses,the Z ring constricts to begin separation of the newly forming daughter cells.FtsZ proteins enable the division of mitochondria in early eukaryotes,but have been lost in animals,fungi and plants.In this study,an FtsZ2 gene?PcFtsZ2?was isolated and identified from P.capsici strain SD33,and the research on the functional characteristics of PcFtsZ2 regulating the development of P.capsici was carried out,and innovative scientific discoveries were obtained,with details as follows:It was clear that PcFtsZ2 can regulate the abnormal division of E.coli cells.Here,we have identified two P.capsici orthologue genes of Escherichia coli FtsZs,PcFtsZ1 and PcFtsZ2.It have been proved that EcFtsZ overexpression can inhibit bacterial cell division.Therefore,in this study,through the heterologous expression of PcFtsZ1and PcFtsZ2,it was found that the expression of PcFtsZ2 and EcFtsZ caused the bacterial cells to divide unnormally and form elongated filamentous cells,which is the same as the overexpression phenotype of EcFtsZ.However,the expression of PcFtsZ1 has less effect on bacterial cell division.This result indicates that,like EcFtsZ,PcFtsZ2 can also inhibit normal cell division.It was proved that PcFtsZ2 can effectively regulate the development and early infection of P.capsici.We measured the expression of PcFtZ2 by qRT-PCR in mycelia,sporangia,zoospores,cysts and germinating cysts and inoculated with P.capsici at different time periods.The results showed that expression levels was higher in the mycelia,sporangia,germinating cyst stages and late infection stage.It was further proved that PcFtsZ2 effectively regulates the growth and development of mycelia and sporangia of P.capsici through silencing/overexpression PcFtsZ2.Here,we analyze the effect of FtsZ2 on the growth and development of P.capsici by silenced and overexpresstion FtsZ2.The results showed that the growth rate of silenced or overexpression transformants is less than that of WT and CK.OPcFtsZ2 produce a less dense aerial hyphae than that of WT and CK.SiPcFtsZ2 lack aerial hyphae.Both SiPcFtsZ2 and OPcFtsZ2 lines produced more abundant branches on the sporangiophores.Therefore we conclude that PcFtsZ2 is necessary for normal mycelial and sporangial development.Mycelial growth and sporangial development phenotypes of PcFtsZ2 silencing and overexpression lines are intensified at low temperatures.SiPcFtsZ2 and OPcFtsZ2 lines were cultured under different temperature condition,We found that their growth rates are significantly slower than WT,CK at 20oand 25oC.The hyphal growth rates of OPcFtsZ2s are close to those of the two controls at 30oC.At 25oC and 30oC,sporangial production of the silenced lines was about 40%of the controls,the sporangia are significantly smaller than the controls and lack the normal apical papillae.At 20oC,however,SiPcFtsZs do not form sporangia at all.At 25oC and 30oC,the shapes of the sporangia produced by OPcFtsZ2s are similar to the two controls.At 20?,OPcFtsZs form fewer sporangia which are significantly smaller,malformed and lack the normal apical papillae.These results show that lower temperatures significantly intensify the phenotypes caused by silencing or overexpression of PcFtsZ2.PcFtsZ2 regulation of mitochondria and actin cytoskeleton and participatation in the pathogenic process of P.capsici.We used Mito-Tracker Green staining to visualize the number and arrangement of mitochondria in PcFtsZ2 silencing and overexpression lines.We found that there are fewer and bigger stained mitochondria in sporangia and mycelia of silenced lines.In the sporangia of overexpresstion lines,stained mitochondria were brighter than in CK and WT.We used rhodamine–phalloidin staining to observe the actin cytoskeleton.We found that plaques of sporangia and hyphae of silenced lines are more numerous than in WT and CK but fewer than in the overexpression lines.In the sporangia of three overexpression lines,the plaques are more numerous,larger,and irregularly shaped relative to controls.In the hyphae of overexpression lines,the more numerous actin plaques are clustered together and more closely positioned.This shows that PcFtsZ2 can regulate the mitochondria and actin cytoskeleton.PcFtsZ2 is essential for pathogenicity.We inoculated with zoospores of SiPcFtsZ2,OPcFtsZ2 in pepper leaves,found that SiPcFtsZ2 significantly reduces germination rates of sporangia,and the sporangia and cysts germination tube are shorter and their pathogenicity are weaker.Although sporangia have a lower germination rate,the sporangia and cysts germination tube of OPcFtsZ2 are longer,which are more pathogenic.This study reveal the the mechanism of RxLR23 induced host immunity and PcFtsZ2regulation of infection,enrich the research theory of pathogenic oomycetes,and provided a theoretical basis for efficient strategies for the prevention and control of P.capsici.