| Cassava（Manihot esculenta Crantz）is an important staple food crop.However,cassava is weakness in basic research and poor in disease resistance.In order to improve cassava study and variety improvement,it is necessary to establish high efficient cassava research method systems and identify stress resistant genes in cassava.Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）is an important enzyme in glycolysis.How more and more studies indicated that GAPDHs were involved in plant immunity and stress response.Thus,we used cassava GAPDH as research object and obtained the research results as follows:1.Two kinds of Agrobacterium tumefaciens AGL-1 and GV3101 were used to over-express green fluorescent protein（GFP）orβ-glucuronidase（GUS）in cassava leaves.The successful examination of GFP and GUS indicating the Agrobacterium tumefaciens mediated transient expression system in cassava leaves was successful establishment.2.Phytoene desaturase（PDS）gene connected to tobacco rattle virus（TRV）silence vector p TRV2.Agrobacterium tumefaciens carrying the targeted vector were injected into cassava leaves.20 days post infiltration,the new grown leaves of cassava appeared albinism,and the albinism area diffused along the vein of petiole to the tip.At the same time,PCR and quantitative PCR was performed to measure silence effect.These results indicated that the VIGS system could be used in cassava.3.The gene numbers and sequence of cassava glyceraldehyde-3-phosphate dehydrogenase（GAPDH）were obtained from JGI database.The evolutionary tree was constructed by GAPDHs protein sequences of rice,Arabidopsis and cassava.From the evolutionary tree,14 GAPDHs of cassava could be divided into 4 groups:cytosolic glyceraldehydes-3-phosphate dehydrogenase（GAPC）,plastid glyceraldehydes-3-phosphate dehydrogenase（GAPCp）,chloroplast glyceraldehydes-3-phosphate dehydrogenase（GAPA/B）and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase（GAPN）.4.The transcript levels of 14 cassava GAPDHs in response of Xanthomonas axonopodis pv.Manihotis（Xam）showed that nearly all 14 GAPDHs could mediate by Xam,and 6MeGAPCs has the most strongest response to Xam.5.The CDS sequence of 6 MeGAPC were fused over-expressed with GFP.Then the subcellular localization of 6 MeGAPCs were found both in cytosolic and nuclear.6.GAPCs silenced cassava plants were constructed and inoculated by Xam,the Xam bacterial number of silenced cassava was less than in mock,indicating MeGAPCs might negatively regulated cassava resistant to cassava bacterial wilt.7.Many interacted proteins of MeGAPCs were screened by yeast two hybrid assay.Then,pull-down and bimolecular fluorescence complementary experiments showed that GAPC4/6 could interact with ATG8b/8e in vitro and in vivo.8.Expression level of MeATG8b/8e and autophagic level were higher than control in MeGAPCs silenced cassava,indicating MeGAPCs negatively regulating MeATG8b/8e.9.GAPDH enzyme activity and transcript level of MeGAPCs were higher in than control in MeATG8b/8e silenced cassava,indicating MeATG8b/8e negatively regulating MeGAPCs.10.GAPDH enzyme activity and autophagic level of MeGAPCs and MeATG8b/8e co-silencing cassava plants were between MeGAPCs silencing cassava plants and MeATG8b/8e silencing cassava,indicating the antagonistic function of MeGAPCs and MeATG8b/8e.11.MeGAPCs silenced cassava showed less Xam contents after inoculated with Xam.Meanwhile,H2O2 and callose were accumulated in MeGAPCs silenced cassava plants indicating the disease resistant phenotype were caused by the activation of innate immunity of cassava plants.12.MeATG8b/8e silenced cassava showed susceptible disease phenotype after inoculated with Xam.Meanwhile,the phenotype of Xam inoculated MeGAPCs and MeATG8b/8e co-silenced cassava plants were similar to control.From these results,MeGAPCs are the negative regulator of cassava bacterial wilt and MeATG8b/8e positively regulated cassava bacterial wilt resistance by repressing MeGAPCs.This study indicates that we established the over-expression system and TRV based gene silence system in cassava,meanwhile,we indicated the important function of MeGAPCs and it’s interacted protein MeATG8b/8e in regulating cassava bacterial wilt resistance.On the one hand,the silence of MeGAPCs could mediate cassava bacterial wilt resistance by activating cassava innate immunity.On the other hand,MeATG8b/8e could also regulate cassava bacterial wilt resistance by regulating MeGAPCs activities.This study illustrated the mechanism of MeGAPCs and MeATG8b/8e regulating cassava bacterial wilt resistance,... |
