Function Of Brassica Napus MPK4 In Resistance To Sclerotinia Sclerotiorum

Sclertinia sclerotiorum (Lib.) de Bary causes a highly destructive disease in oilseed rape, resulting in a tremendous seed yield loss in China and other regions of the world. No immune or highly resistant cultivars of oilseed rape have been reported to date, and few genetic sources of resistance to the pathogen are available to breeders. Control of the disease depends heavily on application of fungicides to the crop, but this is expensive and can be ineffective due to the difficulties associated with applying sprays to thick canopies and a lack of suitable forecasting methods to enable timely application of fungicides. So far, attempts have been made to engineer disease resistance in economically important crop plants. However, the molecular basis of plant defense to this pathogen remains poorly understood, which restricts engineering resistance by transgenic approaches.Plant mitogen-activated protein kinases (MAPKs) have been implicated in plant defense as a new component of the defense signaling pathways. In the study, we isolated a Brassica napus MPK4 (BnMPK4),and over-expressed the gene in oilseed rape to investigate its function in resistance to S. sclerotiorum and its resistance mechanism.A cDNA clone encoding a mitogen-activated protein kinase Brassica napus MPK4 (BnMPK4) was isolated. DNAMAN program analysis show that the entire open reading frame of the cloned cDNA encodes a protein of 373 amino acid residues that exhibit 95%, 85%, 82% and 81% identity with Arabidopsis, tobacco, rice and maize MPK4. BnMPK4 contain conserved amino acid motif (T201E202Y203) and a CD domain in its C-terminal extension. The MAPK alignment using the ClustalX programs indicated that BnMPK4 belonged to B subgroup of The TEY subtype.Using quantitative RT-PCR, we analyzed BnMPK4 expression changes in oilseed rape after treatment with the chemicals BTH, MeJA or oxalic acid. The results showed that BnMPK4 expression were promptly activated by BTH and suppressed by MeJA. The gene expression was promptly activated in OA treatment.Using quantitative RT-PCR, we found that BnMPK4 along with PDF1.2 are inducible in a resistant line Zhangshuang 9, but consistently suppressed in a susceptible line 84039 after inoculation with S. sclerotiorum.To further determine whether BnMPK4 functions in defense responses to the necrotrophic fungus S. sclerotiorum, we constructed a plant transformation vector pG4A-BnMPK4. The cv. 84039 was chosen as the recipient for transformation. Five independent transgenic oilseed rape lines over-expressing BnMPK4 were generated and confirmed by PCR, Southern blot and northern blot. These results showed that the gene BnMPK4 has been inserted into the genome of the recipient and its RNA level was much higher in all five transgenic lines than the untransformed.Transgenic oilseed rape over-expressing BnMPK4 markedly enhanced resistance to S. sclerotiorum. Logistic analysis of lesion development after inoculation showed that enhanced resistance in the transgenic plants would be committed by both delay of lesion occurrence and slow lesion expansion rate. By using trypan blue staining, we found that over-expression of BnMPK4 inhibited the growth of S. sclerotiorum in oilseed rape. Inoculation experiment also indicated that transgenic plants gain enhanced resistance to Botrytis cinerea.To further investigate mechanism of BnMPK4 in Resistance to Sclerotinia Sclerotiorum, analysis of expression changes of defense-marker genes were carried out by quantitative RT-PCR. Results showed high levels of BnMPK4 activity activate PDF1.2 but suppress PR-1 in the absence of induction by chemicals or pathogen treatment. By using 3,3-diaminobenzidine (DAB) staining, we found that transgenic plants constitutively decreased H2O2 production. H2O2 treatment to the transgenic plants resulted in enhanced susceptibility to both S. sclerotiorum and B. cinerea. These results supported the idea that MPK4 positively regulate jasmonic acid (JA)-mediated defense response, which plays an important role in resistance to S. sclerotiorum in oilseed rape.