Study On TaSBT1 In Wheat Resistance Against Stripe Rust

Wheat stripe rust is a prevalent fungal disease caused by the obligate parasitic fungus Puccinia striiformis f.sp.tritici(Pst),which has caused serious economic losses to global wheat production.In order to realize the durable control of wheat stripe rust,it is necessary to find new defense-related genes that can effectively stimulate wheat disease resistance.Previous studies reported that plant subtilases(SBTs)could induce hypersensitive response and systemic acquired resistance in plant-pathogen interactions,indicating that SBTs play important roles in plant disease resistance.However,study on plant SBTs against obligate biotrophic fungal pathogens,especially on Pst,have rarely been reported.Our previous study showed that a wheat subtilase TaSBT1 may be involved in the incompatible interaction between wheat and Pst.However,it is still unclear about the molecular characteristics and the specific functions.In this study,the molecular characteristics and functions of TaSBT1 gene were analyzed by using bioinformatics analysis,subcellular localization,virus-induced gene silencing and qRT-PCR.Our findings can provide theoretical basis for enriching the wheat signal transmission pathway and revealing the molecular mechanism of wheat resistance to stripe rust.The main results are as follows:1.The basic characteristics of TaSBT1: Three homologous of TaSBT1 were cloned and thereafter named as TaSBT1 a,TaSBT1b and TaSBT1 d,which located on chromosomes 4AL,4BS and 4DS of hexaploid wheat cv Chinese Spring(IWGSC RefSeq v1.1),respectively.The open reading frames of these three homologous are 2,304,2,310,and 2,295 bp,and encoded 767,769,and 764 amino acid residues,respectively.Molecular weights of the three protein sequences are 78.47 kD,78.61 kD and 78.25 kD,with theoretical isoelectric points of 5.79,5.99,and 5.71,respectively.It is predicted that TaSBT1 proteins consist of a signal peptide,a prodomain,a subtilisin-like domain,and a protease-related domain.Protein sequences alignment indicates that TaSBT1 closely related to the subtilases of many plants,and shares 99% identity with the homeologous gene of Aegilops tauschii subsp.tauschii.The transient expression of TaSBT1 fused to the green fluorescent protein in the leaves of Nicotiana benthamiana showed that TaSBT1 proteins are distribute outside the plasma membrane,and their secretion function were also confirmed by the yeast signal peptide screen assay successfully.2.The functions of TaSBT1: The expression profiles of TaSBT1 a,TaSBT1b and TaSBT1 d were analyzed by qRT-PCR with wheat leaves infected by Pst.In the incompatible interaction,the transcripts of all three homologous were up-regulated between 12 hour-post inoculation(hpi)and 24 hpi compared with control treatment.The highest expression level of TaSBT1 b was 7.2-fold at 12 hpi compared with the control,while TaSBT1 a and TaSBT1 d were 4.3-fold and 6.2-fold of the control,respectively.The transcripts of the three homologous were also induced significantly(about 2.5-fold)at 24 hpi.In contrast,the expression levels were no significant changes in the the compatible interaction.It is speculated that TaSBT1 may be involved in wheat defense response against stripe rust.Transient overexpression of TaSBT1 in tobacco leaves by Agrobacterium transient expression technique can induce plant cell death,indicating that TaSBT1 protein is involved in programmed cell death.Subsequently,BSMV-VIGS technique was used to silence TaSBT1 in wheat leaves with two specific fragments of TaSBT1 Significant necrosis was observed on the control wheat leaves inoculated with avirulent race CYR20 of Pst,while amount of urediniospores were produced on the gene-silencing leaves.No significant changes were observed in sporulation both in the target gene-silencing plants or the control wheat leaves inoculated with virulent race CYR33.Moreover,necrotic area in gene-silencing leaves stained by trypan blue were less than the control plants.The silencing efficiency of confirmed that the expression levels of TaSBT1 in the silenced plants decreased after inoculation with both avirulent and virulent race.The results suggested that silencing TaSBT1 significantly reduced the resistance of wheat against CYR20.3.Resistance signal pathway that TaSBT1 may be involved in: TaSBT1 was only induced by Salicylic acid(SA)in wheat seedlings after treated by exogenous hormones SA,Methyl Jasmonate and Ethylene,suggesting that TaSBT1 may be involved in SA signal pathway.In addition,STRING prediction results revealed that the proteins with higher possibility of interaction with TaSBT1 were PMEI(AT2G47670),interleukin-I-receptor family protease(AT2G17055),receptor-like kinase BAM1(MPA24.5),MUCILAGE-MODIFIED 2(MUM2)and AAA-type ATPase family protein(AT3G24530).PME activity assay suggested that the protease crude extract from the TaSBT1 gene-silenced leaves showed PME activity that that of control treatment the protease solution extracted from the TaSBT1 gene-silenced leaves showed a lighter color on the agarose gel medium stained by ruthenium red,suggesting a decrease in PME activity.This finding indicated that TaSBT1 could affect wheat disease resistance by regulating wheat PME activity.In summary,the subtilase encoded by TaSBT1,is involved in the incompatible interactions between wheat and avirulent Pst,and regulates the wheat resistance against Pst.This study elucidates the molecular mechanism of subtilases involved in regulating the immune response of wheat to Pst,and provide new strategy for comprehensive control of wheat stripe rust.