Cloning And Pathogenicity Analysis Of The Mulberry Dwarf Phytoplasma Pathogenic-related Protein Genes

Phytoplasmas are wide spread pathogens responsible for a broad range of plant diseases.Mulberry yellow dwarf is one of the most serious infectious diseases caused by phytoplasma,and has lead to large destruction of mulberry and restrict the development of sericultureseriously. Inability to culture phytoplasmas in vitro has hindered their characterization at themolecular level. In this study, two phytoplasma pathogenic-related protein genes were clonedfrom mulberry yellow dwarf phytoplasma by homologous cloning, and the proteins encodedwere analysis with bioinformatic methods, and the pathogenicities of them were also explored.The information obtained in this study will aid to elucidating the molecular mechanisms ofphytoplasma infection and provide a basis for for controlling and curing phytoplasmadiseases.One of the effector proteins of mulberry dwarf phytoplasma was clones and named asMPEP (GenBank accession No. HM153427). The gene was213bp, which was deduced toencode a peptide of70amino acids whose predicted molecular mass was8.19kDa andisoelectric point was5.445. Sequence comparison analysis showed that the MPEP had highesthomology to the same effector proteins from other phytoplasmas. It was predicted that thestructure of MPEP was rich in α-helixes and poor in β-strands, coils and turns. The proteinpossesses three coiled-coil structure and no obvious tertiary structure. The predicted physicaland chemical properties indicate that the MPEP protein possesses an evident signal peptideand a transmembrane domain which overlaps with the N-terminal signal peptide, and showsstrong hydrophilic property, and may be a secretory protein. The coding region of MPEPwithout stop codon was inserted into a prokaryotic shuttle expression vector pBBR1MCS-5including the GFP gene and then was transformed into Bacillus subtilis Lu-144to detect thefluorescence signal of the culture supernatant of Lu-144fermentation broth. The resultsconfirmed that the MPEP was a secreted protein. The coding region of MPEP was insertedinto an expression vector, pET30a(+), and then was transformed into the Escherichia coliBL2l. The gene was successfully expressed in the E. coli BL2l with IPTG induction, and theexpressed protein was found in the culture supernatant which indicated the MPEP was asecreted protein The coding region of the MPEP was inserted into an expression vectorpBI121containing GFP and transformed into Arabidopsis thaliana plants and onion epidermal cells. The roots and protoplasts of the transgenic plants were observed withfluorescence microscopy and the results showed that the MPEP were expressed in thecytoplasm. The coding region of the MPEP was inserted into the expression vector pBI121controlled by the35S promoter and transformed into Arabidopsis thaliana plants. Thetransgenic Arabidopsis thaliana plants showed the symptoms such as witches’ broom,dwarfism, stunted growth and shortened internodes which are typical symptoms ofphytoplasma diseases. The metabolome of the transgenic plants were analysis by GC-MS, andthe results showed that some metabolic components such as sugars, acids and hydrocarbonschanged significantly resulting from the expression of MPEP. All of these results mightindicate that the MPEP is an effector protein of mulberry yellow dwarf phytoplasmas.A full-length DNA encoding hemolysin (designated as MDPH, GenBank accession No.HQ891118) was cloned from mulberry yellow dwarf phytoplasma by homologous cloning.The gene was717bp, which was deduced to encode a peptide of238amino acids whosepredicted molecular mass was27.3kDa and isoelectric point was9.29. Sequence comparisonanalysis showed that the MDPH had highest homology to the hemolysins from otherphytoplasmas. It was predicted that the structure of MDPH was rich in α-helixes, β-strandsand coils, and was poor in turns. The predicted physical and chemical properties indicate thatthe MDPH protein possessed some evident hydrophobic and hydrophilic regions which couldform7transmembrane helixs. The protein possesses evident antigenicity regions, but nosignal peptide, and was a non-classical secretory protein. The coding region of MDPH wasinserted into an expression vector pET30a(+) and then was transformed into the E. coli BL2l.The gene was successfully expressed in the E. coli BL2l with IPTG induction. The plantexpression vector pBI121-MDPH was transformed into Arabidopsis thaliana plants, and thetransgenic plants some phenotypes such as smaller pods and lower tough and magenta stems.The results in this study may be helpful to study the functions of MPEP and MDPH, andlay a foundation for elucidating the pathogenic mechanism of mulberry yellow dwarfphytoplasma.