The Mechanism Of Calcium Signal Regulating Plant Responses To Bemisia Tabaci

Bemisia tabaci（Gennadius）（Hemiptera:Aleyrodidae）is an agricultural pest distributed worldwide.Bemisia tabaci threatens crop production and causes huge damage by direct feeding on leaves and indirectly transmitting plant virus,such as tomato yellow leaf curl virus（TYLCV）.Among the biotypes of B.tabaci,B and Q are the two most invasive and destructive biotypes.Calcium signature refers to the temporal and spatial changes of calcium ion(Ca²⁺)concentration in plant cells in respond to external stimuli.It is one of the earliest signal events in the process of plant recognition of external stress,and plays a very important role in triggering downstream signaling pathways and regulating plant defense.Plant-mediated interactions between virus and insect vectors are known to exert important influences on the epidemiology of plant diseases and the abundance of herbivores.The role of plant defense in mediating the tripartite interaction of plant-virus-vector has been reported to be regulated by phytohormones,but whether this interaction is mediated by early calcium signaling remains unclear.We conducted this research based on the invasive whitefly B.tabaci and the devastating begomovirus TYLCV and two important host plants Arabidopsis and tomato.The research content explored the previous overlooked aspect in plant-virus-vector interaction.The results reveal the difference in the plant early defense calcium signal induced by viruliferous and non-viruliferous whitefly,and the mechanism of plant defense against to B.tabaci regulated by the calcium signal.This study not only expands our understanding of the sophisticated interaction between begomovirus and its insect vector,and promotes the research on the regulatory network of plant-induced defense,but also provides new hints for the control of vector and virus spread.The main results of this study are as follows:（1）we used the ultrasensitive fluorescent calcium biosensors（GCa MP6 and aequorin）in combination with viruliferous and non-viruliferous B.tabaci B and Q to study real-time calcium dynamics in vivo.Ou results found that infestation of viruliferous and non-viruliferous B.tabaci B and Q triggered the increase of he cytosolic Ca²⁺concentration([Ca²⁺]_cyt)in GCa MP6 Arabidopsis leaf regions around and systemic to the feeding site,and viruliferous B.tabaci significantly reduced the[Ca²⁺]_cyt elevations.Moreover,TYLCV infection of B.tabaci significantly reduced[Ca²⁺]_cyt elevations and transmembrane Ca²⁺influx in tomato expressing aequorin that experienced actual and simulated herbivory by B.tabaci.This is the first report that plant virus impact host plant-insect vector interaction by suppressing plant early Ca²⁺signaling.（2）The transcriptome of tomato leaves fed by viruliferous and non-viruliferous B.tabaci B and Q was determined by using second-generation sequencing technology.The analysis of transcriptome data revealed that the infestation of viruliferous and non-viruliferous B.tabaci B and Q induced different gene expression patterns in tomato plants,and the differentially expressed genes up-regulated by viruliferous and nonviruliferous B.tabaci were mainly involved in calcium signal pathway.Bemisia tabaci feeding on tomato leaves significantly induced the up-regulation of candidate calcium signal genes Sl CML35 and Sl CDPK29 by q RT-PCR,indicateing that Sl CML35 and Sl CDPK29are two calcium signaling genes in tomato plants in respond to B.tabaci.（3）Sl CML35 was significantly induced by B.tabaci feeding.The tomato mutants with function loss of CML35 was constructed by using CRISPR/Cas9.It was found that the function loss of CML35 in tomato plants significantly reduced the plant resistance to whitefly.The survival rate,fecundity and development rate of whitefly B.tabaci on the slcml35 tomato mutants were significantly higher,suggesting that CML35 is a positive defense regulator.The lower expression of jasmonic acid（JA）synthesis genes and JA response genes,and the lower levels of JA and JA-Ile in slcml35 plants underlie their impaired defense against whitefly B.tabaci.（4）Sl CDPK29 was significantly induced by B.tabaci feeding.The tomato mutants with function loss of CDPK29 was constructed by using CRISPR/Cas9.The loss of function of CDPK29 led to an increasing resistance of plants to B.tabaci.The growth rate of B.tabaci on the slcdpk29 tomato mutant was significantly reduced.After B.tabaci feeding,the tomato mutants slcdpk29 was significantly induced the up-regulation of JA synthesis genes and down-regulation of salicylic acid（SA）synthesis genes.B.tabaci feeding significantly increased the JA-Ile and reduced SA,resulting in slcdpk29 plants were more resistant to whitefly,showing that CDPK29 is a negative regulator of plant defense against to B.tabaci.（5）Calcium signal contributing to JA-induced direct and indirect defense against the whitefly B.tabaci in tomato plants.Exogenous JA not only increases the[Ca²⁺]_cyt in tomato mesophyll cells but also induces the expression of Ca²⁺-sensor genes and plant defense genes,thus decreased the fitness of B.tabaci on JA-treated plants.JA treatment also significantly increased the release of plant volatiles,which significantly reduced the feeding preference of B.tabaci to JA-treated plants,and attracted the wasp of B.tabaci.Pretreatment with Ca²⁺inhibitor significantly repressed the elevation of[Ca²⁺]_cyt,expression of JA-induced genes and emission of plant volatiles induced by JA,thus reduced the JA-induced direct and indirect plant resistance to B.tabaci.