Analysis of the Relationship between Blast Resistance Genes and Disease Resistance of Rice Germplasm via Functional Molecular Markers

Rice blast disease is one of the most devastating diseases of rice(Oryza sativa L.)caused by the fungus Magnaporthe oryzae(M.oryzae),and neck blast is the most destructive phase of this illness.The underlying molecular mechanisms of rice blast resistance are not well known.Thus,we collected 150 rice varieties from different ecotypes in China and assessed the rice blast resistances under the natural conditions that favoured disease development in Jining,Shandong Province,China in 2017.Results showed that 92(61.3%)and 58(38.7%)rice varieties were resistant and susceptible to M.oryzae,respectively.Among the 150 rice varieties screened for the presence of 13 major blast resistance(R)genes against M.oryzae by using functional markers,147 contained one to eight R genes.The relationship between R genes and disease response was discussed by analysing the phenotype and genotype of functional markers.The results showed that the rice blast resistance gene Pita was significantly correlated with rice blast resistance.Our results provided a basis for the further understanding of the distribution of 13 major R genes of rice blast in the germplasm resources of the tested rice varieties,and were meaningful for rice disease resistance breeding.

Development of Hybrid Rice Variety FY7206 with Blast Resistance Gene Pid3 and Cold Tolerance Gene Ctb1

Hybrid rice Fanyou 7206（FY7206）, derived from the cross between a sterile line Fanyuan A and a restorer line Fuhui 7206, was bred by the Rice Research Institute, Fujian Academy of Agricultural Sciences, China. FY7206 was characterized by moderate blast resistance, cold tolerance, as well as wide adaptability, and high yields. The blast resistance results indicated that the frequencies of blast races in race B, race C and the total resistance frequency for FY7206 were 95.5%, 100.0% and 97.2%, respectively. The disease resistance results showed that the leaf blast grade for FY7206 was level 1 and panicle blast was level 5. The indoor spray results indicated that FY7206 was resistant to 11 isolates of Magnorpathe oryzae. The blast resistance of FY7206 might be derived from the high expression of blast resistance gene Pid3. The results for simulated cold resistance in an artificial climate chamber indicated that the cold tolerance for FY7206 was moderate at the booting and flowering stages. The cold tolerance results also indicated that FY7206 could be tolerant to temperatures as low as 10 °C at the seedling stage. The q RT-PCR results showed that the expression of cold tolerance gene Ctb1 in FY7206 was relatively high. These results suggested that FY7206 is a hybrid indica rice variety with good comprehensive characteristics, including blast resistance and cold tolerance.

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2, conferring gene-for-gene resistance to the Chinese blast strain ZB15, was isolated from a rice variety （Digu） by the map-based cloning strategy. Here, we constructed a control plasmid pZH01-pi-d2tp309 （pZH01-tp309） and three different expression constructs, pCB-Pi-d25.3kb （pCB5.3kb）, pCB-Pi-d26.3kb （pCB6.3kb） and pZH01-Pi-d22.72kb （pZH01-2.72kb） of Pi-d2, driven by Pi-d2 gene’s own promoter or CaMV35S promoter. These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu, Taipei 309, Nipponbare and Zhonghua 9 through Agrobacterium- mediated transformation. A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin. PCR, RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited. Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15. Transformants exhibited resistance to rice blast at various levels. The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference. The own promoter of Pi-d2, about 2.2 kb or 3.2 kb, had the similar promoter function as CaMV35S. Field evaluation for three successive years supported the results of artificial trial, and some lines with high resistance to rice leaf blast and neck blast were obtained.

Effect of exogenous salicylic acid on resistance of rice seedlings to blast

Salicylic acid(SA)is an endogenous regulatorymolecule for plant flowering and thermogene-sis,and has been reported as a signal moleculeof plant disease resistance in the plant-pathogen interaction.Exogenous SA is capableof inducing disease resistance in a wide range ofplants.This study was to evaluate the poten-tial for SA to induce resistance of rice seedlings

New technique for identifying varieties resistance to rice blast

After 8 yrs lab experiments and field tests, an advanced technique for identifying varieties resistance to rice blast was developed by a research group in Plant Protection Institute, Zhejiang Academy of Agricultural Sciences. With this technique, the inoculum was prepared on a maize-rice-straw-agar media which was suitable for sporulation of most rice blast pathogen isolates. To avoid possible virulence variation during the vegetative growth, the conidia were harvested and preserved for later different sets of resistance evaluation tests. A simple and precise new method—smear inoculation was used for infection. The experi-

Studies on durable resistance of rice to blast in different latitudes

It has been an important problem on resistantbreeding that cultivar’s resistance of rice toblast disease caused by Magnaporthe grisea(Hebert)Barr.is easily broken down.In orderto find out resources with durable resistance toblast,the study was carried out during 1990--

Pyramiding of Pi46 and Pita to improve blast resistance and to evaluate the resistance effect of the two R genes

Utilization of R（resistance） genes to develop resistant cultivars is an effective strategy to combat against rice blast disease. In this study, R genes Pi46 and Pita in a resistant accession H4 were introgressed into an elite restorer line Hang-Hui-179（HH179） using the marker-assisted backcross breeding（MABB） procedure. As a result, three improved lines（e.g., R1791 carrying Pi46 alone, R1792 carrying Pita alone and R1793 carrying both Pi46 and Pita） were developed. The three improved lines had significant genetic similarities with the recurrent parent HH179. Thus, they and HH179 could be recognized as near isogenic lines（NILs）. The resistance spectrum of the three improved lines, which was tested at seedling stage, reached 91.1, 64.7 and 97.1%, respectively. This was markedly broader than that of HH179（23.5%）. Interestingly, R1793 showed resistance to panicle blast but neither R1791 nor R1792 exhibited resistance at two natural blast nurseries. The results implied that the stacking of Pi46 and Pita resulted in enhanced resistance, which was unachievable by either R gene alone. Further comparison indicated that the three improved lines were similar to HH179 in multiple agronomic traits; including plant height, tillers per plant, panicle length, spikelet fertility, and 1 000-grain weight. Thus, the three improved lines with different R genes can be used as new sources of resistance for developing variety. There is a complementary effect between the two R genes Pi46 and Pita.

RICE TRANSFORMATION WITH A PHYTOALEXIN GENE AND BIOASSAY OF THE TRANSGENIC PLANTS

Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.

An OsPRMT5-OsAGO2/miR1875-OsHXK1 module regulates rice immunity to blast disease

Rice ARGONAUTE2(OsAGO2)is a core component of the rice RNA-induced silencing complex(RISC),which is repressed by Magnaporthe oryzae(M.oryzae)infection.Whether and how OsAGO2-mediated gene silencing plays a role in rice blast resistance and which sRNAs participate in this process are unknown.Our results indicate that OsAGO2 is a key immune player that manipulates rice defense responses against blast disease.OsAGO2 associates with the 24-nt miR1875 and binds to the promoter region of HEXOKINASE1(OsHXK1),which causes DNA methylation and leads to gene silencing.Our multiple genetic evidence showed that,without M.oryzae infection,OsAGO2/miR1875 RISC promoted OsHXK1 promoter DNA methylation and OsHXK1 silencing;after M.oryzae infection,the reduced OsAGO2/miR1875 led to a relatively activated OsHXK1 expression.OsHXK1 acts as a positive regulator of blast disease resistance that OsHXK1-OE rice exhibited enhanced resistance,whereas Cas9-Oshxk1 rice showed reduced resistance against M.oryzae infection.OsHXK1 may function through its sugar sensor activity as glucose induced defense-related gene expression and reactive oxygen species(ROS)accumulation in Nipponbare and OsHXK1-OE but not in Cas9-Oshxk1 rice.OsAGO2 itself is delicately regulated by OsPRMT5,which senses M.oryzae infection and attenuates OsAGO2-mediated gene silencing through OsAGO2 arginine methylation.Our study reveals an OsPRMT5-OsAGO2/miR1875-OsHXK1 regulatory module that fine tunes the rice defense response to blast disease.

A natural allele of Bsr-d1 in rice confers broad-spectrum blast resistance

Subject Code:C14With support from the National Natural Science Foundation of China,a collaborative research group led by Prof.Chen Xuewei(陈学伟)of the Rice Research Institute,Sichuan Agricultural University,published an article entitled“A natural allele of a transcription factor in rice confers broad-spectrum