Functional Analysis Of MeCIPK23-MeCBL1/9 Interaction In Cassava Defense Response Against Xanthomonas Axonopodis Pv.Manihotis

Cassava is one of the three major potato species in the world.It is widely cultivated in tropical and subtropical regions and is the main food crop for low-income people in the tropics.Cassava bacterial blight is a serious disease that endanger the yield of cassava.So far,there are only a few genes which have been reported involved in disease resistance in cassava.Therefore,it is of great significance to develop cassava resistance-related genes to improve cassava yield.Calcium ions play essential roles in plant development and stress signaling pathways.Calcineurin B-like proteins(CBLs)and CBL-interacting protein kinases(CIPKs)are crucial components of calcium signals.In this study,systematic expression profile of 25MeCIPKs in response to Xanthomonas axonopodis pv.manihotis(Xam)infection was examined,by which 7 candidate MeCIPKs were chosen for functional investigation.These 7 genes were transiently expressed in tobacco leaves,and the disease-resistance-related gene was screened for functional analysis.The selected disease-resistant gene and MeCBLl and MeCBL9 were silenced in cassava using virus-induced gene silencing(VIGS).Then we detect the change of disease resistance in cassava with Xam infiltration.Based on our experiments,we obtained the following results.First,systematic expression profile of 25 MeCIPKs in response to Xanthomonas,axonopodis pv.manihotis(Xam)infection was examined,seven MeCIPK genes(MeCIPK5,MeCIPK8,MeCIPK12,MeCIPK22,MeCIPK23 and MeCIPK24)were selected for functional analysis.Then,the coding regions of the 7 MeCIPKs were connection into GFP in pEGAD vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens strain GV3101 and infiltrated into Nicotiana henthamiana leaves,green fluorescent and cell nuclei was then visualized using a confocal laser-scanning microscope,it was found that these 7 genes are located in the nucleus and cell membrane.Second,we found over express the 7 MeCIPK.s can affects the transcripts of defense-related genes and can induce callus deposition in tobacco leaves at the injection site,In addition to MeCIPK7,all of the other 6 MeC’IPKs had an inhibitory effect on.Xam.Third,we found MeCIPK23 could interact with MeCBL1 and MeCBL9 respectively through the yeast two-hybrid and BiFC assay.Over expressing MeCBL1 and MeCBL9 in tobacco could improve the defense ability of tobacco.In contrast,inhibiting MeCIPK23,MeCBL1 and MeCBL9 in cassava had the same effect.In conclusion,we found MeCIPK23 as well as MeCBLl and MeCBL9 that confer enhanced defense response against Xam,and the result can provide potential genes for breeding new varieties cassava which has disease-resistant.