Map-Based Cloning And Functional Characterization Of Spotted-Leaf Gene SPL40 In Rice(Oryza Sativa L.)

Spotted leaf mutants show typical necrotic lesions that appear spontaneously in the absence of any pathogen attack.These mutants are often characterized to exhibit programmed cell death（PCD）and activation of plant defense responses resulting in enhanced disease resistance to multiple pathogens.Here,we reported a novel spotted-leaf mutant,spl40 which was derived from the EMS mutagenesis of indica rice cultivar Zhongjian-100.We carried out phenotypic characterization,measurement of agronomic traits,physiological and biochemical characterization,detection of cell death occurrence and ROS accumulation,evaluation of disease resistance,genetic analysis and mapping of the mutation,functional complementation and subcellular localization.This study provide the evidence for enhanced disease resistance response and physiobiochemical changes exhibited by spl40,along with its genetic characterization.The results obtained in this study are as follows:1.Initially lesions appeared at leaf tips during seedling stage and gradually covered the whole leaf at the tillering stage.The lesion development was light-dependent.spl40 showed obvious cell death at and around the lesion,and burst of reactive oxygen species（ROS）was accompanied by disturbed ROS scavenging system.Photosynthetic capacity was compromised as evidenced by significant reductions in chlorophyll content,important photosynthesis parameters and downregulated expression of photosynthesis-related genes which ultimately led to poor performance of major agronomic traits.2.spl40 exhibited enhanced resistance to 14 out of 16 races of bacterial blight pathogen of rice,caused by Xanthomonas oryzae pv.oryzae,most probably though activation of SA and JA signaling pathways,owing to upregulated expression of SA and JA signaling genes,though the exact mechanism remain to be elucidated.The results obtained here clearly show that genes related to defense and PCD were upregulated in accordance with enhanced disease resistance and occurrence of PCD,whereas the photosynthetic capacity and overall ROS homeostasis was compromised in spl40.3.The spotted-leaf phenotype was controlled by a novel single recessive nuclear gene.Genetic mapping combined with high throughput sequencing analysis identified Os05G0312000 as the most probable candidate gene.Sequencing of ORF revealed a single SNP change from C to T that resulted in non-synonymous change in amino acid residue from leucine to phenylalanine.Functional complementation study using WT allele showed that complementation plants developed lesion mimic phenotype during later stages of plant growth.4.An allelic mutant spl40^NIP was identified with mutation in the same locus but exhibited different phenotype than spl40.At2g48110 is the ortholog of SPL40 and Arabidopsis mutant,reduced epidermal fluorescence 4（ref4）with mutation in At2g48110 was originally reported to be recessive,but plants heterozygous for ref4 exhibited intermediate growth and phenylpropanoid-related phenotypes,suggesting that these mutations are semidominant.We are considering that the phenotype is dose-dependent and the locus may possess complicated biological function possibly associated with genotypes because of the diverse phenotypes.To further clarify this enigma,we are carrying out another complementation by transferring mutant allele into wild type background as well the genetic interaction between spl40 and spl40^NIP.5.Subcellular localization study showed that SPL40 is localized to the cytosol.SPL40 was found to express in most of the plant parts and at different plant growth stages.Overall,our data suggest that a novel spotted-leaf mutant,spl40,would help to elucidate the mechanism behind lesion development and its involvement in programmed cell death and associated defense responses.