Isolation And Functional Analysis Of Receptor-like Kinase Genes TaRLCK1B And TaCRK10

In the process of plant growth and development,the plantsare often endangered bya complex and variedset of stresses.Receptor-like protein kinasesare receptors and signal componentsin plant responses to developmental signals and various kinds of stresses,and play important roles in plant defense responses to stresses and development.In this study,two receptor-like protein kinases,TaRLCK1B and TaCRK10,which are involved in resistant responses to wheat sharp eyespot,were isolated from wheat by RNA sequencing(RNA-Seq),comparative transcriptome and real-time quantitative PCR(RT-qPCR),and defensive functions of both geneswere analyzed by virus-induced gene silencing(VIGS).The aim of this study is to lay a foundation for early stage research on molecular breeding of forest disease resistance.The results as follow:(1)TaRLCK1B protein,encoded byTaRLCK1B,is a cytoplasmic receptor protein kinase composed of 404 amino acids.RNA-Seq sequencinganalysis and RT-qPCR results showed that the transcriptional level of TaRLCK1B in resistant wheat varieties was significantly higher than that in susceptible wheat varieties.The gene transcription level in wheat resistant varieties was significantly increased after infection of the pathogen.The results of phylogenetic tree and protein sequence alignment analyses showed that the TaRLCKlB protein was closely related to OsRLCK176(with 84.03%identity)and related to BIK1(with 61.14%identity).Through Virus-induced gene silencing following assessment the wheat response to Rhizoctonia cerealis,the resultsindicated that the silencing of TaRLCK1 weakened resistance of the host plants to Rhizoctonia cerealis.Meantime,silencing of TaRLCK1 significantly elevated both the content of H2O2and the transcriptional level of the(Reactive oxygen species,ROS)-generating enzyme-encoding gene RBOH,but repressed the expression of the ROS-scavenging enzyme-encoding gene CAT1 at 18 hours after inoculation with R.cerealis.These results suggest that TaRLCK1B contributes to wheat resistance to Rhizoctonia cerealis by regulating early ROS signals.(2)TaCRK10 protein,encoded byTaCRK10,is a cysteine-rich receptor-like protein kinase comprised by 591 amino acids.TaCRK10 expressionis associated with resistancedegreeof wheat cultivars againstRhizoctonia cerealis,Subcellular localization of TaCRK10in wheat protoplast indicated that the TaCRK10 protein was localized to the cell membrane.In wheat treated byplant-hormones,the transcriptional level of TaCRK1O gene was analyzed,and the results showed that TaCRK1O gene was induced by jasmonic Acid(JA)and Aminocyclopropanecarboxylic acid(ACC).After VIGS experiment,the expression of TaCRK1O gene was decreased termed silencing,and the disease test indicated that silencing of TaCRK10 gene weakened the resistance of host wheat plants to Rhizoctonia cerealis The results of RT-qPCR analysis showed that the transcription levels of Chitinase3 and Chitinase4 were significantly reduced in the TaCRK10-silenced wheat plants.Taken together,the resultssuggest that TaCRK10ipositively regulates the expression levels of Chitinase3 and Chitinase4 and wheat resistance to Rhizoctonia cerealis.