Application Of Loop-Mediated Isotheral Amplication(LAMP)in Rapid Detection Of Phytophthora Infestans And Screening Of Novel Plant Pattern-Recognition Receptor

Potato is one of the four most important staple crops in the world.China is the most potato production in the world,accounting for about a quarter of the world yield.Late blight caused by Phytophthora infestans is a devastating disease that can cause great economic losses in production,leading to one billion dollars of direct annual economic losses in China.The ability to sensitively detect and accurately diagnose the cause of a disease is a crucial step towards effectively managing it.To this end,many DNA-based diagnostic tools have been developed for P.infestans.Although these methods can detect the disease accurately,it will take a long time to diagnose the disease.Currently existing methods for P.infestans detection require,at minimum,thermal cycling and gel electrophoresis equipment.In some cases,more expensive quantitative fluorescence detection equipment is required,so these methods might not be suitable for developing countries.Therefore,there is a growing demand for simple and economical molecular tests.Loop-mediated isothermal amplification(LAMP)is an attractive alternative to conventional DNA-based diagnostic techniques because of its minimal equipment requirements,sensitivity,specificity and ability to produce rapid results.Based on a LAMP assay and using the newly target gene PiA3aPro,we designed and screened a set of highly specific and sensitive primers allowing amplification at 64? over 70 min.In the validation assay,detection of 15 strains of P.infestans isolates from around the world,the results found were showed positive,but many other Phytophthora,Pythium,fungal and bacterial strains still were negative.Found in the sensitivity test,the system can detect the minimum concentration of 10fg·?L-1 DNA of P.infestans.The PiA3aPro-LAMP assay rapidly and successfully diagnosed P.infestans in field samples,its sensitivity for detecting the pathogen in soil was 10 zoospores in 0.25g soil.To summarize,this study reports the development of a LAMP assay targeting the PiA3aPro element for visual detection of P.infestans.The PiA3aPro-LAMP assay efficiently amplified the target element in less than 70 min at 64? with high specificity and sensitivity,and it can significantly shorten the detection time and reduce testing costs.These results suggest that the PiA3aPro-LAMP assay reported here shows great potential for the visual detection of P.infestans in plants and production fields.At present,resistant cultivars and fungicides are two main stratigies to prevent late blight.However,due to the rapid propagation and evolution of the pathogen,the resistance of crop varieties and the effect of chemical control are very easy to lose,so more sustainable methods are required.One way to improve plant disease resistance is to enhance the capability of the plants,own innate immune system.As an important part of plants' defense against pathogens,host can recognize microbial invaders by detecting pathogen-associated molecular patterns(PAMPs)by cell surface pattern-recognition receptors(PRRs).Many studies suggested that heterologous expression of PAMP recognition systems could be used to engineer broad-spectrum disease resistance to important bacterial pathogens,potentially enabling more durable and sustainable resistance in the field.XEG1 is a widespread and conserved PAMP found in the Phytophthora,which belongs to glycosyl hydrolases family.XEG1 can trigger defense responses including cell death.Nevertheless,the mechanisms that allow plants to perceive pathogens and to defend against infection remain only partly know.LRRs(leucine-rich repeats)family contains a conserved leucine repeat sequence,which is the largest class of PRRs.It can be divided into two categories:receptor-like kinase(Receptor-Like Kinases,RLKs)and Proteins(Receptor-Like),according to the difference of extracellular region.In previous work,over400 receptors were identified in Nicotiana benthamiana belonging to the LRR family.This study involved in the construction of LRR receptor silencing vector library,and successfully constructed 105 LRR receptor silencing vectors.VIGS screening revealed that XEG1 and its homologous genes in the TRV:R3 treated N.benthamiana can not induce hypersensitive reaction.The TRV:R3 silenced two homologous LRR receptors,named RXEG1A and RXEG1B,respectively.These two receptors may be involved in the recognition of XEG1.Then we prove that only the receptor-like protein RXEG1A can interact with XEG1 through Co-IP.In addition,we found that RXEG1A can interact with XEG1 in intercellular space through Pulldown.Furthermore,PsXEG1 can interact with RXEG1A LRR motif.Subsequently,we overexpress RXEG1A in N.benhamiana,which can suppress pathogen infection.In this study,we identified a receptor like protein RXEG1A,which provides a new material for breeding and improvement of potato late blight resistance.