Studies On The Mechanism Of SWEET Sugar Transporters In The Parasitic Process Of Meloidogyne Incognita

Meloidogyne incognita parasitize many crops and cause significant economic losses worldwide.However,the control measures of this disease are limited.It is of great significance to gain the effective measures to control the M.incognita base on mechanism of M.incognitaplant interaction.SWEETs(sugars will eventually be exported transporters)has the function of bidirectional sugar transport and participates in the interaction between various pathogenic microorganisms and plants.In this study,We analyzed SWEET gene expression of Arabidopsis thaliana and soybean(Glycine max.)through real-time fluorescent quantitative PCR(q PCR),GUS and YFP experiments after the inoculation with root-knot nematode(RKN).Then,26 interacting proteins of At SWEET1 were screened out from the c DNA library of M.incognita by yeast one hybrid(Y1H)and analyzes its function.The specific research results are as follows:1.SWEET genes relative expression of A.thaliana and soybean: There are 17 SWEET sugar transporters in A.thaliana.Through the staining experiment,we find out that the nematodes were moving towards feeding sites in A.thaliana at 6 days post inoculation(dpi).At 12 dpi,nematodes developed to the stage of Sausage-J2 and developed to the stage of Globose at 18 dpi.According to results of q PCR,At SWEET4,At SWEET6 and At SWEET12 genes were induced at 1 dpi and the relative expression level of At SWEET4 was the highest,12.87 times higher than that of the control group.At 3 dpi,At SWEET2,At SWEET3,At SWEET5,At SWEET10 and At SWEET12 were induced and the relative expression of At SWEET2,At SWEET5 and At SWEET12 were induced at 6 dpi.At 12 dpi,the expression levels of the SWEET sugar transporter genes in A.thaliana were down-regulated except At SWEET1 and At SWEET15.At 18 dpi,the expression levels of At SWEET1,At SWEET3,At SWEET4,At SWEET6,At SWEET9 and At SWEET10 were up-regulated.There are 52 SWEET sugar transporters in soybean.Through the staining experiment,the nematodes began to invade the soybean roots at 1 dpi and to migrated within the nematode roots at 3dpi.At 6 dpi,nematodes had thickened and established the feeding sites.And we can see the knot on the root surface meanwhile.At 12 dpi,nematodes developed to the stage of Sausage-J2 and developed to the stage of Globose at 18 dpi.We can see that the male adult nematodes appeared in the root at 24 dpi and the nematodes were leaving the root at 30 dpi.According to results of q PCR,the relative expression levels of SWEET genes in the root of soybean were changed in the different times.At the same time,the relative expression levels of SWEET genes in Stem,Leaf,Flower,Bean and Pod were detected through q PCR.The expression levels of 35 genes were varied in different degrees.2.The infection and development of nematodes in A.thaliana root: The infection and development of nematodes in A.thaliana atsweet10a?atsweet11a?atsweet12b?atsweet13a?atsweet14a?atsweet15d?atsweet11a12b?atsweet12b15d and atsweet11a12b15 d mutants root were analyzed at 18 dpi by the staining experiment.In the roots of mutant plants,the proportion of J2 and Sausage-J2 was higher than that of the control group,while the proportion of Globose was lower than the control group.The number of galls in the mutant plants atsweet10 a,atsweet12b,atsweet15 d,atsweet12b15d and atsweet11a12b15 d was significantly different(p<0.01)from that in the control group,while the number of galls in atsweet11 a and atsweet11a12b15 d was significantly different(p<0.05)from that in the control group.The GUS staining and Laser Scanning Confocal Microscopy(LSCM)experiments showed that the At SWEET11 a,At SWEET 12 b and At SWEET15 d genes fused with GUS and YFP protein were significantly expressed in galls.3.The screening of the proteins in M.incognita which interact with the promoter of At SWEET1 gene: 26 yeast monoclones were screened out through yeast one hybrid.Finally,11 sequences were obtained which were interact with the promoter of At SWEET1 gene.Finally forecasted that Mi-7?Mi-8?Mi-10 and Mi-19 may be the secrete proteins using the online software Signal P-5.0(http://www.cbs.dtu.dk/services/Signal P/),TMHMM 2.0(http://www.cbs.dtu.dk/services/TMHMM/)and database Worm Base(https://wormbase.org/#012-34-5).In conclusion,the relative expression of SWEET sugar transporter genes of A.thaliana and soybean were influenced after those hosts were infected by RKNs,and were significantly increased in the tissues where forming the feeding sites.It was predicted that related proteins in M.incognita interacted with the promoter of At SWEET1 of A.thaliana gene and involved in the interaction of M.incognita-A.thaliana pathosystem.