Identification And Functional Analysis Of Conserved Target CAD7 By Phytophthora Avr3a Family Effectors

Oomycetes are eukaryotic microorganisms that resemble fungi in morphology and are close to brown algae in evolution.Plant pathogenic oomycetes cause devastating crop diseases resulting in huge economic losses.The crop blight caused by P.infestans,P.sojae,and P.capsiciare serious threats to the sustainable production of potato,soybean,pepper,and so on.Phytophthora secrete hundreds of sequence-divergent RXLR effectors,which play important roles in the pathogenic processes.RXLR effectors could enter into the host plant cells to interfer their normal physiological and biochemical functions through modifying or manipulating the host targets,thus facilitating pathogen colonization.Therefore,identification and functional analysis of the effector targets is key to dissect the molecular basis of host-Phytophthora interaction.Avr3a-like effectors(also calledAvr3 a family effectors)are conserved in Phytophthora and are present in P.infestans,P.sojae,and P.capsici.For example,both the two well-known RXLR effectors PiAvr3 a and PsAvr1 b belong to this family.We found that ArabidopsisCinnamyl Alcohol Dehydrogenase 7(CAD7)can interact with Avr3 a family effectors.In this study,we focused on identification and functional analysis of CAD7,which is the conservative host target of Avr3 a family effectors.Our main discoveries are as follow:(1)In Arabidopsis-P.capsici,N.benthamiana-P.capsici and N.benthamiana-P.infestans pathosystems,we confirmed that both AtCAD7 and NbCAD7(the ortholog of AtCAD7 in N.benthamiana)could interact with multiple Avr3 a family effectors through yeast two-hybrid(Y2H),bimolecular fluorescence complementation(BiFC),and Co-immunoprecipitation(Co-IP)assays.(2)Genome-wide analysis of CAD genes revealed that the CAD7 subfamily proteins are highly conserved and fast evolving.Real-time PCR analysis indicated that the expression levels of CAD family genes in Arabidopsis are differentially expressed in response toP.capsiciinfection.Semi-quantitative PCR analysis and promoter activity analysis further confirmed that AtCAD7 subfamily genes were induced by both P.parasitica and P.capsici infections.These results demonstrate that the AtCAD7 subfamily genes are involved in plant defense to Phytophthora and suggest that they were most likely to be targeted by Avr3 a family effectors.(3)AtCAD7silenced Arabidopsis line(Ri7)was prepared by RNA interfere(RNAi)technology.Inoculation of Ri7 plant showed that silencing of AtCAD7 decreased colonization by P.parasitica.NbCAD7-silenced N.benthamianaplant(tNb7)was prepared by virus induced gene silence(VIGS)technolgy.Infection assays showed that silencing of NbCAD7 decreased the colonization of P.parasitica,P.capsici,and P.infestans.Consistently,ovexpression of AtCAD7 in Arabidopsis enhanced its susceptibility to P.parasitica and P.capsici.These results indicate that CAD7 is a negative regulator of plant defense to Phytophthora.(4)Cell death assays in tNb7 plants showed that silencing of NbCAD7 specifically promoted PAMP(pathogen associated molecular pattern)INF1-induced plant cell death.And overexpression of NbCAD7 in N.benthamiana attenuated plant cell death induced by INF1.Thus we conclude that NbCAD7 can suppress INF1-triggered cell death.(5)The analysis of R3a-Avr3 a,Rb-Avrblb1,R1-Avr1 and Rx-CPtriggered cell death in tNb7 plants indicated that silencing of NbCAD7 could specifically suppress R3a-Avr3 a triggered cell death.In addition,Y2 H and BiFC experiments showed that CAD7 could interact with R3 a weakly.These results indicate that CAD7 is specifically involved in R3a-Avr3 a recognition.(6)BiFC assays showed that neither of the AtCAD7 mutants for the three domains could interact with Avr3 a family effectors while both of the two AtCAD7 mutants for ciritical residues could still interact with Avr3 a family effectors,suggesting that the three conserved domains(Zn1-binding domain,Zn2-binding domain and NADPH-binding domain)in AtCAD7 are required to the interaction while the ciritical residues are not.(7)NbCAD7-FLAG was coexpressed with different Avr3 a family effectors in N.benthamiana and the protein accumulation of NbCAD7-FLAG was detected through Western blot at 3 days after injection.The result showed that NbCAD7-FLAG was stabilized by Avr3 a family effectors at different degrees.Taken together,we show that plant cinnamyl alcohol dehydrogenase 7(CAD7)is a conserved target of Avr3 a family effectors in Phytophthora.CAD7 is a negative regulator of plant defense.It not only suppresses INF1 triggered cell death,but also is involved in R3a-Avr3 a recognition.During Phytophthora infection,CAD7 is up-regulated in both transcriptional level and protein level.The Avr3 a family effectors are responsible for the stabilization of CAD7 to promote infection.