The Study On Mechanism Of Tomato MiR394 In Resistance To Phytophthora Infestans

Tomato(Solanum lycopersicum)is vulnerable to a variety of pathogens,and it is an ideal specie for studying the pathogenic mechanism of plants.Late blight is a devastating disease of tomato,which can cause severe yield reduction.Conventional prevention and control methods have some limitations,so it is particularly important to use genetic engineering methods to discover key genes and cultivate disease-resistant varieties.Non-coding RNA(nc RNA)in the genome plays a crucial role in the response of plants to biological stress.Nc RNA includes micro RNA(mi RNA),long non-coding RNA(lnc RNA),small interfering RNA(si RNA),and many other types.mi R394 is a member of the mi RNA family,and its expression level changed significantly after pathogen infection.Our previous study predicted three lnc RNAs(lnc RNA40787,lnc RNA27177,and lnc RNA42566)that contain mi R394 endogenous target mimics(e TM),which may exist as the competitive endogenous RNAs(ce RNAs)of mi R394.Based on the expression pattern among these lnc RNAs and mi R394,lnc RNA40787 was chosen for further investigation.Subsequently,mi R394 and lnc RNA40787 were overexpressed and silenced,respectively,to clarify the mechanism and function of mi R394 and lnc RNA40787 in tomato resistance against late blight.First,to explore the role of mi R394 in tomato resistance against late blight,our study constructed the overexpression plasmid by ligating it with the efficient plant expression vector p BI121.Short tandem target mimic(STTM)was used to silence mi R394.Two recombinant vectors were transformed into the tomato by transient infection.We found that the overexpression of mi R394 could reduce the transcription level of its target gene Leaf Curling Responsiveness(LCR),and affect the expression level of jasmonic acid(JA)related genes.These ultimately reduced tomato resistance to late blight.The negative regulatory role of the mi R394 was demonstrated.Secondly,after obtaining the sequences of these three lnc RNAs(lnc RNA40787,lnc RNA27177,and lnc RNA42566)that related to mi R394,the e TM sites on these lnc RNAs were analyzed.Furthermore,these lnc RNAs showed a clear upward trend,while mi R394 was significantly inhibited,and the LCR expression pattern was roughly the same as lnc RNA40787.For further exploration,we overexpressed lnc RNA40787.After transiently overexpressed lnc RNA40787,the expression level of mi R394 decreased significantly,and the expression level of LCR increased accordingly.These results proved that mi R394 can be decoyed by lnc RNA40787,thereby regulating the transcription level of target gene and affecting the resistance of tomato to late blight.Finally,the silencing and mutation vector of lnc RNA40787 were constructed.After lnc RNA40787 was silenced,the expression of mi R394 increased significantly,and the expression of LCR decreased.After inoculated with Phytophthora infestans,the diameter of lesion on tomato leaves increased significantly.To illustrate the direct interaction between lnc RNA40787 and mi R394,the mutation vector of lnc RNA40787 was constructed,and it was found that the expression of mi R394 did not change significantly after the transformation of mutant lnc RNA40787.Based on the above results,we can conclude that mi R394 can indeed be decoyed by lnc RNA40787.In summary,this study validated the existence of the ce RNA-lnc RNA40787 of mi R394 and found the negative regulatory effect of mi R394 after the infection of Phytophthora infestans.Our research provides information on the lnc RNA40787-mi R394-LCR regulatory network,providing a theoretical basis and reference for revealing the molecular mechanisms of tomato resistance against late blight.