The Function Analyses Of Solanaceous Small G Proteins ROPs To Phytophthora Pathogens

Phytophthora pathogens can infect more t han one hundred of plant species resulting in destructive diseases.To control the diseases caused by these notorious pathogens,cultivating qualified resistant cultivars is urgently needed.To support the breeding process,it is i mportant to confirm the physiological race type of Phytophthora pat hogens.It is also i mportant t o screen novel resistant genes and unravel the resistant mechanisms to assist the breeding strategy.In this study,we first identified the pathogens isolated from diseased pepper plants as Phytophthora capsici based on morphology and molecular biology.And we confirmed the physiological race type of P.capsici based on a set of international differential hosts.To select the resistant pepper parental lines,we screened the pepper germpl asm resources by root inocul ating zoospores of P.capsi ci on pepper.In the mean ti me,the Solanaceous small G proteins were identified by using genome-wide screening.And the proteins were classified based on phylogenetic analysis.The results as followed:1.Seven identidied P.capsici strains varied in biological characteristics.P1 isolated from Guangdong was identifi ed as race 2 and other six isolates,P2 to P7,were identified as race 3.2.The resistant level of 27 pepper parental lines were measured by root inoculation of zoospores of P.capsici.Three lines were identified as highly resistant level,6 lines were identified as resistant level,3 lines were identified as moderate resistant level and 15 lines were identified as susceptible level.3.To evaluate the chemical drug resistance risk of P.capsici,we measured the myceli um growt h inhibition rate of fluazinam and cyazofami d on 7 P.capsici isolates.The results showed that the mycelium growth inhibi ted by fluazinam varying from 42.68 to 71.85 %,whereas inhibited by cyazofamid varying from 1.87 to 55.01 %.Cyazofamid had t he lowest inhibition rate on P5,which was only 1.87%.The results indicated that some P.capsici isolates had chemi cal drug resistance risk against cyazofami d at different levels.4.To identify the small G proteins ROPs in Solanaceous famil y,we performed genome-wide screening on Solanum tuberosum,Solanum lycopersicum,Solanum melongena,Capsi cum annuum,Nicotiana benthamiana and Nicotiana tabacum using 11 ROPs of Arabidopsis thaliana as input.The results showed that the number of ROPs i n S.tuberosum,S.l ycopersicum,S.melongena,C.annuum,N.bent hamiana and N.tabacum was 16,9,5,8,13 and 14,respectively.In total,we found 65 ROPs in these 6 Solanaceous plant species.And the phylogenetic analyses showed that 65 ROPs were classifi ed as 5 clades and proposed as Clade ?,?,?I,? and ?.5.To confirm the function of Clade ? in Solanaceous plant species to P.capsici,we first measured the expression level of Sl ROP-?.1 at different inoculating time points of P.capsici by real-time quantitative PCR.The results showed that the expression level of Sl ROP-?.1 was highly induced at 36 hpi,which was 4.6 ti mes of 0 hpi.In the mean ti me,we silenced Nb/Sl/Ca ROP-?.1 using the same vector of virus-induced gene silencing.And we measured plant resistant level to P.capsici after 4 weeks of TRV treatment.The results showed that the Nb/Sl/Ca ROP-?.1 silenced plants were more susceptible to P.capsici compared with TRV: GUS treated plants.6.To confirm wheather Solanaceous ROP-? have broad-spectrum resistant to different Phytophthora pathogens,we measured the expression level of St ROP-?.2 at different inoculating time poi nts of P.infestans by realtime quantitative PCR.The results showed that the expression level of St ROP?.2 was hi ghly induced at 6 hpi,which wa s 4.4 ti mes of 0 hpi.Overexpression of St ROP-?.2 in S.tuberosum(potato)could enhance the resistance to P.infestans.And transi ent expression of St ROP-?.2 and CASt ROP-?.2 in N.benthamiana could promote the resistance to P.c aps ici.The concentration of salicylic acid was elevated in overexpressing potat o lines of St ROP-?.2 and CASt ROP-?.2.The concentration of SA and the expression level of Nb ROP?.1 were significantly decreased in N.benthamiana t ransiently expressed with Nah G codi ng salicylate hydroxylase.In the mean time,N.benthamiana plants were more susceptible after transiently expressing of Nah G.The results indicated that Solanaceous small G protein ROP-? is involved i n Phytophthora resistance by mediating SA signalling pathway.