Improvement Of Rice Resistance And Plant Architecture By Editing Pi21 And D3 With CRISPR/Cas9 Technology

As a populous country,ensuring the stability of grain production is an important issue related to the national economy and people's livelihood.In view of limited land resources,cultivating multi-resistant,high-yield and high-quality rice varieties is the fundamental way to ensure food security.Traditional methods such as hybrid breeding have been unable to meet the increasing breeding objectives because of the unclear molecular mechanism,poor targeting and time-consuming problems.On the contrary,CRISPR/Cas9 technology is highly efficient and precise by editing plant endogenous genes to improve resistance and increase yield.YH208?Indica?has the advantages of high yield and good quality,but it is susceptible to rice blast.Previous studies showed that the resistant pi21 allele had a consistent effect against all 10 of widely distributed race of M.oryzae.DS?Japonica?has the characteristics of cold tolerance and good quality,but its plant height is too high,lodging,tillering is less and other adverse traits.D3 gene is reported to negatively regulate tiller and plant height.Therefore,we used CRISPR/Cas9 to introduce a loss-of-function mutation into the Pi21 and D3 gene in two elite varieties YH208 and DS,respectively.On the one hand,we hope to improve their resistance and plant architecture;on the other hand,it provides material basis for further exploring the mechanism of Pi21 and D3 and rice genetic improvement.1.Using CRISPR/Cas9 to edit the Pi21 gene to improve the resistance of YH208.In this work,we screened a restorer line YH208 with high susceptibility to panicle blast,and comparative analysis of the Pi21 allele in YH208 with the Pi21 susceptible allele,resulting in a C-to-T substitution and 9,15bp deletion at 238-247 in YH208.It is different from the reported susceptible genotypes,so we speculate that it may be a new susceptible allele.We designed a double target site on the second exon of Pi21,and a total of 10 positive line were obtained by genetic transformation and screening.Through analysis,they were divided into three mutation types:one homozygous mutation and two heterozygous biallelic mutations.Further,20 mutants containing“transgenic-free”were screened from the T1 generation.2.In summer field conditions,we found that the pi21 mutant appeared lesions mimic on the lower?older?leaf tips and showed short plant height.Until the flowering lesions mimic phenotype completely disappears.To determine whether the development of lesions in loss-of-function Pi21 involves altered H₂O₂ accumulation and cell death,we performed 3,3 C-diaminobenzidine?DAB?and Trypan blue staining,The result show that a large amount of hydrogen peroxide accumulation and cell death in the pi21 mutant leaves.Meanwhile,we have further studied the transcription level of PR genes using qRT-PCR.PR1a,PR10b and WRKY45,OsLOX2,EIN2,which are involved in the SA,JA,EHT signaling pathway.The results show that these genes were significantly up-regulated in pi21.3.The wild type of YH208 was punch inoculated with a total of 46 race of M.oryzae.We screened 7 affinity strains from the inoculation experiment.To test pi21 mutant resistant against M.oryzae.We selected TJ13 and DZ108 to punch inoculate pi21 mutant that cultivated in greenhouse and field environments.Punch inoculation assays revealed that lesion length were significantly lower in lesion mimic pi21 mutant than in YH208.At the same time,we were inoculated with Xoo isolates P3 and Rhizoctonia solani,and the results showed that the pi21mutant was susceptible to sheath blight,but did not affect the resistance of bacterial blight.4.According to the statistical analysis of the main agronomic traits of pi21 mutant,the results shown that pi21 mutant did not affect rice quality,tiller number and grain number per panicle,but has influence on plant height and seed setting rate,especially the yield is significantly lower than the wild type.5.Using CRISPR/Cas9 to edit the D3 gene to improve plant architecture of DS.Firstly,we performed a sequence alignment of the D3 allele in DS with the D3 allele of the negatively regulated tiller in Nipponbare.And the sequence was found to be identical,indicating that the D3 gene in DS also negatively regulates tillering.We designed a single target site at the front end of the D3 gene exon.Four types of homozygous mutants were obtained from the T1generation,all of which were base deletions.Among them,KO#1 line is deletion 3bp resulting in no frameshift mutation.Further,51 mutants containing“transgenic-free”were screened from the T2 generation.6.According to the leaf age statistics found that the d3 mutant growth period was shortened by one week compared to the wild type.Through two consecutive generations of investigations,it was found that the agronomic traits of T1 homozygous mutants can be stably inherited to offspring.Further investigation found that except for the agronomic traits of KO#1 line,there was no significant difference compared with the wild type.The other three lines showed dwarf and increased tillers,but shorter panicle length and fewer grains per panicle,which resulted in no difference in final yield.7.By inoculating the d3 mutant with Magnaporthe oryzae,Xanthomonas oryzae pv.oryzae and Rhizoctonia solani,the results showed that the d3 mutant enhanced resistance to the bacterial blight compared with the wild type,but hyper-susceptibility to sheath blight and rice blast.Compared with the wild type,the d3 mutant showed a significant down-regulated of defense-related gene expression after inoculation with M.oryzae,and a decrease in hydrogen peroxide accumulation at the invasion site.In summary,we used the CRISPR/Cas9 to edit the Pi21 gene of YH208 and the D3 gene of DS,pi21 mutant showed spontaneous cell death and enhanced resistance to rice blast,the d3mutant showed increased tillering,dwarf,improved plant architecture,and enhanced resistance to bacterial blight.In addition,we obtained 20 and 51“transgene-free”mutants that were homozygous of pi21 and d3.There“transgene-free”mutant may provide materials for more in-depth research functions and the molecular mechanism of resistance response in rice.They will also contribute to rice breeding and reginal introduction.