Map-Based Cloning And Functional Analysis Of OsCNGC9 Gene Controling Immunity And Functional Analysis Of Two Transcription Factors Controlling Heading Date In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)feeds more than half of the world's population.Plant diseases cause significant crop loss,which is a serious threat to global food security.For a long time,a large number of pesticides have been used as an important means to control diseases.However,with the increasing environmental pollution and people's increasing demand for green and healthy foods.Molecular design breeding of disease-resistant using resistance genes has become the important direction of rice disease-resistant breeding.The core of molecular design breeding of disease-resistant is the identification of resistance genes.During the perpetual battle with pathogens,plants have developed a two-tiered innate immune systems:pathogen-associated molecular pattern(PAMP)triggered immunity(PTI)and effector-triggered immunity(ETI).PTI is generally considered to confer plants broad-spectrum disease resistance,and ETI isconfer plantsrace-specific resistance.In practice,dominant resistance(R)genes confer strong and race-specific resistance,which are usually not durable and ineffective for new pathogen races with a few years.Therefore,the identification of broad-spectrum disease-resistance genes has more important value for rice disease-resistant breeding.However,due to the technical limitations of identification and breeding,only a few rice broad-spectrum resistance genes and their molecular mechanisms have been reported.The study for a novel lesion mimic mutant showed that OsCNGC9-mediated Ca2+influx triggered defense responses during PTI.Furthermore,activated SA signal-transduction cascade upregulated the transcriptional level of OsCNGC9,thereby enhancing the broad-spectrum disease resistance of rice.These results provided new ideas to further elucidate the molecular mechanism of rice PTI.In addition to reducing the yield losses caused by external diseases,enhancing the ability of rice to use light and temperature in the environment is also an important measure to ensure stable or increased rice yield.The heading date is a transition point between vegetative growth and reproductive growth and is closely related to rice yield.We screened dominant gain-of-funciton transcriptional factor(TF)population(pubi::TFs-VP64)and found that the two transcription factors OsMYBIR1 and OsHAPL1 can increase rice yieldby delaying rice heading date.Main results are as follows:1.We identified a novel lesion mimic mutant cdsl.Cytological and transcriptional level analysis showed significant lesion mimic and enhanced cell death marker gene strongly expression occurred in leaves of mutant plants after heading date.The identification of disease resistance revealed that cdsl plantswere more susceptible to rice blast in seeding stage.Genetic analysis revealed that the lesion mimic phenotype of mutant plants was controlledby a single recessive nuclear gene.Through map-based cloning,the target gene was mapped to a 40-kb genomic region.Nine putative open reading frames were predicted in this mapping region.Sequence analysisrevealed that a 4-bp deletion was occurred in the coding region of OsCNGC9.This deletion error was predicted to introduce a premature stop codon.Genetic complementation assay in mutant revealed that the lesion mimic phenotype and reduced disease resistance of complementary transgenic plantsdisappeared.Using the CRISPR/Cas9 technology to knock out the endogenous OsCNGC9,the phenotypes of lesion mimicand reduced disease resistance inknock-out transgenic plants were reproduced.2.OsCNGC9 is a Ca2+-permeable divalent cation-selective inward channel and is involved in regulating rice PTI responses.Expression analysis showed that the endogenous OsCNGC9 was expressed in all tissues examined,with relatively higher expression in mesophyll cells of leaves.Analysis of Protein Properties revealed that OsCNGC9 is localized to the plasma membrane and can form homologous or heterologous complexes with itself and other members of the rice CNGC family.Ion permeability and selectivity analysis showed that OsCNGC9 functions as an inward Ca2+ channel,and the mutation in OsCNGC9 disrupted its calcium channel activity.The OsCNGC9-mediated Ca2+ influx is essential for PTI-induced signaling events.Leaves of cds1 were less sensitive to fungal chitin and bacterial flagellin.After chitin or flagellin treatment,the influx rate of calcium ions in mesophyll cells,the accumulation of reactive oxygen species and the expression levels of defense marker genes were significantly lower than that of wild type.Further analysis showed that the PTI-triggered SA signal-transduction cascade was impaired in cdsl,and SA induced the transcription of OsCNGC9.There are two WRKY transcription factor binding sites in the OsCNGC9 promoter,OsWRKY45 promotes OsCNGC9 transcription by directly binding to the W-box-2 motif in OsCNGC9 promoter.In addition,up-regulation of OsCNGC9 transcription increases broad-spectrum blast resistance.3.OsMYB1R1 and OsHAPL1 increase rice yield by delaying heading date.By screening dominant gain-of-funciton transcriptional factor(TF)population,we identified two new transcription factors,OsMYBIRl and OsHAPL1,that regulate the heading date of rice,both of which can delay the heading date of rice variety "Kitaake" and increase rice yield.Further analysis showed that they all delayed flowering by inhibiting the expression of Ehdl and the downstream florigenous genes.Both OsMYBIR1 and OsHAPL1 expression patterns are constitutive and rhythmically controlled.Among them,OsHAPL1 can also form complexes with DTH8 and Hdl to regulate rice heading date under long-day conditions.In addition,our results also indicate that the modification of crop genes by using the transcriptional activator VP64 may be a potential method for crop genetic improvement.