Map-based Cloning And Functional Analysis Of Rice Lesion Mimic Gene SPL33

Plant lesion-mimic mutants?LMMs?spontaneously develop localized cell death lesions resembling those caused by hypersensitive response?HR?in the absence of pathogens,and most of LMMs display significantly enhanced resistance to diseases.It is believed that such mutants are ideal tools for deciphering the signal pathways of PCD and defense responses in plants.Here we isolated and characterized a new rice lesion-mimic mutant named spotted leaf 33?spl33?.The SPL33 was identified through a map-based cloning strategy combined with sequencing,and predicted to encode a eukaryotic translation elongation factor one alpha?eEF1A?-like protein.The loss-of-function of SPL33 leads to the lesion mimic phenotype and enhanced disease resistance.Results of our experiments are summarized as follows:1.The spl33 mutant started to develop small and reddish-brown lesions on the leaves at three-leaf stage and continued to develope till the ripening stage.The phenotypes of spl33remained stable in different experimental sites and years.The mutant spl33 developed spontaneous necrotic lesions and spreaded cell necrosis through all growth stages.Besides,the lesion mimic phenotype of spl33 mutant was induced by light and temperature.2.Genetic analysis revealed that the spl33 phenotype was caused by a single recessive nuclear gene mutation.We mapped SPL33 gene to a 70 kb region on telomere of chromosome 1 with 476“mutant×Dular”F₂ recessive individuals.There are 11 open reading frames?ORFs?in this region.Sequence analysis showed that 4^th ORF?LOC_Os01g02720?had a single-base substitution(G²⁴⁹³?T²⁴⁹³)in its 7^th exon,leading to a premature stop,which resulted in the loss-of-function of SPL33.Genomic complementary and overexpression test demonstrated that LOC_Os01g02720 is SPL33.3.SPL33 was predicated to encode an eEF1A-like protein consists of 655 amino acid,containing a zinc finger domain which is not present in other eEF1As,and a GTP-binding domain and two oligonucleotide binding domains.Dissection of SPL33 by transformation indicated that the C-terminal region of SPL33 containing the three EF-Tu domains is enough for functioning,whereas the zinc finger domain is redundant,at least for preventing rice leaves from showing lesion mimics.Transcriptome sequencing of spl33 and WT showed that SPL33 functions in cell death,disease response,photosynthesis and biotic stimulus in addition to protein translation.SPL33 was ubiquitously expressed in all organs and growth stages,and SPL33 protein is localized to the ER.4.3,3'-diaminobenzidine?DAB?and trypan blue staining showed that mutant spl33 formed spontaneous necrotic lesions accompanying with H₂O₂ accumulation;TUNEL assay showed that cell necrosis in spl33 leaves was a type of programmed cell death?PCD?;Aniline blue staining displayed that a lot of callose was accumulated in the mesophyll cells of spl33.Loss of chlorophyll,breakdown of chloroplasts,down-regulationofphotosynthesis-relatedgenes,andup-regulationof senescence-associated genes suggested that early leaf senescence happened in spl33 plants.5.The PR genes were all significantly up-regulated in spl33 plants during the development of lesion mimics,and enhanced resistance to blast and bacterial blight pathogens were observed in the spl33 mutant.Transcriptome sequencing analysis suggested that the plant-pathogen interaction pathway was constitutively activated in the spl33 mutant,and most of the differentially expression genes involved in PAMP-triggered immunity?PTI?and effector-triggered immunity?ETI?were significantly up-regulated.These results suggested that defense responses were activated in spl33,resulting in broad-spectrum resistance to pathogens.6.The yeast two-hybrid assays showed that SPL33 protein could interact with 26S proteasome subunit OsRpt3 and OsRpn11,which suggested that SPL33 involved in proteasome-mediated degradation of unfolded and damaged proteins.SPL33 also interacted with LMMs gene RLS1 and OsLMS,which suggested that loss-of-function of SPL33 resulted in broken interactions.These will establish the foundation for the further study of SPL33 functions.