Genetic Determinants On Geminivirus Satellite DNA Inducing Vein Thickening And Enation Symptoms

Begomovirus-DNAβdisease complexes induce different sets of symptom phenotypes in their natural hosts.Tobacco curly shoot virus(TbCSV) and its DNAβinduces leaf curling in Nicotiana benthamiana,N.tabacum cv.Samsun-nn,N. glutinosa and Solarium lycopersicum,while Tomato yellow leaf curl China virus (TYLCCNV) and its DNAβinduces distinctive vein thickening and enations as well as leaf curling.To investigate the genetic determinants of the phenotypic differences, we inoculated Nicotiana spp.with infectious clones of TbCSV/DNAβand TYLCCNV/DNAβpseudorecombinants and showed that symptoms co-segregated with the DNAβcomponent.DNAβencodes an open reading frame(ORF)βC1 on the complementary-sense strand,which is conserved among different molecules in terms of position and sequence.Mutational analysis has shown that theβC1 of TYLCCNV DNAβplays an essential role in symptom induction.Our previously study has been showed that transgenic N.benthamiana and N.tabacum plants expressing TYLCCNV DNAβC1 developed pleiotropic morphologic abnormalities such as leaf curling,distortion and interveinal protuberances on the abaxial surface. To determine whether theβC1 protein of TbCSY is also responsible for symptom induction,we prepared transgenic N.benthamiana and tomato plants expressing the TbCSV DNAββC1.Of the rooted plantlets,only 10%of transgenic N.benthamiana plants were moderately abnormal,including upward leaf curling.Transgenic tomato plants developed normally and remained symptomless.We further investigated the contributions ofβC1 and a 430 nt fragment containing the A-rich region and a 173 nt or 200 nt fragment upstream of the translation start site of theβC1 gene(referred to AP) to phenotypic differences by constructing hybrid satellites in which the DNAββC1 coding region or the AP was exchanged,respectively.Hybrid satellites were inoculated onto their common hosts using either TbCSV or TYLCCNV as helper viruses.A TYLCCNV DNAβhybrid having TbCSV DNAββC1 lost the ability to elicit the vein thickening and enation phenotypes.On the other hand,TbCSV DNAβ hybrids containing TYLCCNV DNAβpC1 or the AP fragment failed to induce the characteristic vein thickening and enations.A TYLCCNV DNAβhybrid having the TbCSV AP fragment produced the enations,but the number of enations was less and their size was reduced.We found that a full length fragment upstream of theβC1 gene of TbCSV DNAβhas a promoter activity and confers a constitutive GUS expression pattern in transgenic tobacco plants by histochemical localization and fluorimetric determination of GUS activity.Southern blot analysis revealed that all of the hybrid satellites were able to infect plants systemically and that the accumulation of either the helper virus or the satellite was not correlated with the phenotypic differences. The above results indicate that DNAβ-encodedβC1 protein has a major contribution to symptom differences between TbCSV/DNAβand TYLCCNV/DNAβbut that theβC1 promoter can also have influence on the symptom production.N.benthamiana plants were inoculated by PVX vector fused with theβC1 gene of TYLCCNV DNAβand the plants inoculated by PVX as the negative control.RNAs of these two samples were extracted used for miRNA microarray analysis.The results indicate that miRNAs homologous with miR398b in cottonwood,miR397 in tomato, miR397a and miR166a in Arabidopsis were significant different between the two samples.Northern blot analysis confirmed that the level of miR165/166 homolog was reduced in N.benthamiana plants expressed theβC1 gene of TYLCCNV DNAβ.It suggests that theβC1 protein may induce development abnormalities by perturbation of miRNA-mediated function.