| Lesion mimic mutants were spontaneously produced lesions in leaves,stems and other parts of plants without any adversity,mechanical damage and pathogen infection,similar to the necrotic spot produced by pathogen infection.Studies have shown that most lesion mimic mutants can enhance disease resistance during the process of necrotic spots.Therefore,lesion mimic mutants are ideal materials for studying programmed cell death and disease resistance mechanism in plants.A lesion mimic mutant spl41 was identified from indica rice Huang Huazhan mutated by ethyl methyl sulfonate(EMS).Analysis showed that the 523 rd nucleotide upstream of LOC_Os01g25450 gene was mutated from G to A,and the loss of function caused by single base mutation leads to lesions phenotype.By phenotype identification,physiological and biochemical analysis,resistance screening and promoter activity analysis,the main results are as follows:The spl41 mutant began to exhibit reddish-brown spots at three-leaf stage and gradually spread to the whole leaves with the growth and development.Compared with the wild type,the plant height,panicle length,tiller number,1 000-grain weight and seed setting rate of spl41 mutant were significantly decreased.The photosynthetic pigment content of spl41 mutant decreased with the number of lesions increased.The assimilation rate,transpiration rate,stomatal conductance and water use efficiency of mutant spl41 were lower than those of wild type,which indicated that the formation of necrotic spots affected photosynthesis.Transmission electron microscopy showed that the spl41 chloroplast was gradually degraded,the arrangement of thylakoid was disordered and reduced in number,which suggested that leaf senescence happened in spl41 plants.Trypan blue staining exhibited that there were a large number of dead cells in spl41.NBT and DAB staining showed accumulation of H2O2 and O2-around spl41 lesions.The enzyme activities of SOD,CAT and POD in spl41 leaves were higher than that of wild type.These results indicated that there was a large accumulation of ROS in spl41.Aniline blue staining indicated that there was a large accumulation of callose in spl41 mesophyll cells.The mutant spl41 significantly increased resistance to rice bacterial blight,rice blast and bacterial leaf streak.q RT-PCR analysis showed that the expression levels of PR1b、PR3、PR4、PR5、PBZ1 and Cht1 were up-regulated in mutant spl41.These results indicated that defense response were activated in spl41.p1391z-spl41promoter-GUS and p1391z-WTpromoter-GUS vectors were constructed to transform Arabidopsis and rice by Agrobacterium-mediated method to explore the expression activity of spl41 promoter.GUS staining showed that the 2020 bp region of spl41 promoter could drive the expression of subsequent gene.The promoter and partial sequence of coding region of spl41 was cloned for functionally complementation of lesion mimic phenotype by using Agrobacterium mediated transformation method.The results showed that the spl41 promoter plus 4800 bp coding sequence was not the complete sequence of the mutant to produce reddish-brown spot.Next,it is necessary to extend the cloning of spl41 gene sequence to determine the functional region responsible for lesions phenotype.Taken together,these results exhibited that spl41 was accompanied by premature senescence of plants and the outbreak of reactive oxygen species during the generation reddish-brown spots,and could significantly improve the resistance of rice to bacterial blight,bacterial streak and blast.The spl41 promoter can drive the expression of the GUS gene,laying a foundation for the cloning of lesions gene and unraveling the molecular mechanism of disease resistance. |
PDF Full Text Request