Functional Characterization Of A Spotted-leaf Mutant HM143 In Rice

Spotted-leaf mutants are ideal materials for evaluating the mechanisms underlying disease resistance and programmed cell death（PCD）in rice（Oryza sativa L.）because they exhibit hypersensitive responses similar to pathogen infection-induced through mediated programmed cell death,which plays a crucial role in plant immunity by initiating the cell death process to protect against pathogen attack.To attain more insight into the function of spotted-leaf mutants in programmed cell death and plant immunity,we carried out the functional analysis of a spotted-leaf mutant,originally termed as HM143,which was isolated from ethyl methane sulfonate（EMS）mutagenesis of an indica rice cultivar IR64.Our findings are summarized as follows:1.Under natural summer field conditions in Hangzhou,China,the mutant leaves displayed abnormal brown spots three weeks after sowing which lasted through the entire growth period.The performance of essential agronomic traits was practically similar between the HM143 mutant and wild type IR64 aside from seed setting rate and 1000-grain weight which were remarkably reduced in the mutant.At the same time no differences were observed in panicle length,plant height,grain number per panicle and panicle number per plant.The results indicated that the mutation caused negative effects to some agronomic important traits.2.The development of necrotic lesions was associated with a deficiency in chlorophyll metabolism and photosynthesis as evidence of decreased chlorophyll content levels,and photosynthetic capacity parameters such as net photosynthetic rate,stomatal conductance,and transpiration rate were decreased in HM143 plus lowered expression levels of photosynthetic related genes and up-regulation of chlorophyll biosynthetic genes.These results indicated that the mutation impaired rice chlorophyll metabolism and photosynthesis.3.Programmed cell death was activated in HM143 compared to WT as we detected a higher accumulation of H₂O₂,positive TUNEL nuclei staining,and up-regulation of PCD-related genes.Besides,the POD,SOD,and CAT activities for the reactive oxygen species（ROS）scavenging system of HM143 were notably higher than those of WT.The contents of MDA and H₂O₂,which are both cell death indicators,were also significantly higher in the mutant than the wild type.The results suggested that cell death in HM143 was mainly due to the over-accumulation of hydrogen peroxide.4.PCD was accompanied by elevated resistance to bacterial blight（BB）pathogen,Xanthomonas oryzae pv.oryzae（Xoo）.The pathogenesis-related（PR）genes including EDS1,NPR1,PR1b,PR10,PR1a,PR4,PR2,PAL4,WRKY82,WRKY85,WRKY45,JAMYb,LOX,PBZ1,CHS1,JAZ8,JAZ6,and PAD4 were significantly activated in the mutant compared to WT.The results demonstrated that enhanced disease resistance to BB was owing to the increased expression of defense response genes in HM143.5.The spl^HM143mutation was fine mapped to the long arm of chromosome 4 with a physical distance of 176 kb between RM18662-RM18668 markers.The candidate region is composed of 20 open reading frames（ORFs）.After sequencing,we found out that the 14^th ORF（LOC＿Os04g25400）was the candidate gene because it has a single base substitution in its second nucleotide of 12^th intron,leading to alternative splicing.Functional complementation of LOC＿Os04g25400 using the WT allele in the mutant background ultimately rescued the spotted-leaf phenotype,indicating that LOC＿Os04g25400 was most likely the target gene responsible for the spotted-leaf phenotype of HM143.6.LOC＿Os04g25400 contains 16 exons,and 16 introns,and encodes a putative7-hydroxymethyl chlorophyll a reductase（HCAR）,a highly conserved enzyme involved in chlorophyll biosynthesis in plants.The rice HCAR（Os HCAR）consists of309 amino acid residues,and is constitutively expressed in all rice tissue tested.Subcellular localization experiment revealed that Os HCAR localizes to chloroplasts.7.To understand the function of Os HCAR in rice,we generated Os HCAR knockout lines using the CRISPRE/Cas9 technology.The resultant Os HCAR knockout-lines displayed WT-like spotted-leaf phenotype,exhibited leaf senescence,and programmed cell death.More importantly,they displayed increased disease resistance to 5 Xoo races,coupled with upregulation of pathogenesis-related（PR）genes.Alternatively,the transgenic lines overexpressing Os HCAR in Kitaake background conferred bacteria blight susceptibility to the 5 races of bacterial blight pathogen in accompany with lowered expression levels of PR genes.These results further confirmed that Os HCAR was responsible for the spotted-leaf phenotype and regulated disease response by activating the expression of PR genes in association with hydrogen peroxide signaling in rice.In conclusion,our results revealed that the mutation in Os HCAR was responsible for the spotted-leaf phenotype of HM143.We speculate that the lesion formation led to a deficiency in chlorophyll and photosynthetic capacity,activated PCD,and over-accumulation of ROS,which ultimately trigged the enhanced disease resistance response.