Enhanced Rice Blast Resistance By TALENs And CRISPR/Cas9 Genome-editing Techniques

Rice is one of the most important food crops in the world, feeding nearly 50% of the world’s population. Rice blast, caused by the filamentous ascomycete fungus Magnaporthe oryzae (asexual generation:Pyricularia oryzae), is one of the most destructive diseases affecting rice in all rice-growing countries and often causes serious damage to rice production. Enhancing the resistance of rice to M. oryzae has been shown to be the most economical and effective approach for controlling rice blast. In conventional rice breeding, it takes approximately a decade or longer to pyramid multiple blast resistance genes into a rice variety via crossing and backcrossing, while the high pathogenic variability in M. oryzae often leads to the rapid break down of resistant cultivars.RNAi-based down-regulation of rice blast disease-susceptibility genes or disease-susceptibility related transcription factor genes expression has been demonstrated to be an alternative approach for enhancing rice resistance to blast. However, expression of RNAi transgenes varies in different transgenic lines, and hard to obtain high-expression transformant lines. In addition, rice plants derived by RNAi methods are usually regarded as transgenic and are subjected to rigorous regulatory processes. Compared with RNAi, TALENs and CRISPR/Cas9 have no such defects; which can precise and high-efficiency targeting specific gene knockout, and obtain the mutant plants harboring the desired gene modification but not containing the transferred DNA by segregation, resulting in no security concerns of genetically modified (GM) food.Rice OsERF922 encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by both virulent and avirulent pathovars of M. oryzae. OsERF922 is localizes in the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. OsERF922 acts as a negative regulator of blast resistance.This thesis consists of two parts. Part Ⅰ:Reseach of whether AvrXa23-based TALENs can target modification of OsERF922; Part Ⅱ:utilizing CRISPR/Cas9 genome-editing techniques targeting modification of OsERF922 to enhance rice blast resistance, and also analysis of detail characteristics via CRISPR/Cas9-targeted. The main results are as follows:1. Three pairs of AvrXa23-based TALENs (T-KJ5/KJ6, T-KJ7/KJ8 and T-KJ9/KJ10) were designed for targeting OsERF922. The gene-editing activity of T-KJ5/KJ6 and T-KJ7/KJ8 have not been detected in rice protoplasts, while T-KJ9/KJ10 exhibits gene-editing activity in rice protoplasts, calli and To plants; what’s more, the T-KJ9/KJ10-targeted somatic mutagenesis frequency of 15.0% and 2.73% in rice calli and To plants, respectively; indicating that the AvrXa23-based TALENs system can be used for site-specific genome editing in rice. Additionally, all types of the mutations were significantly different from the wild-type in PCR amplicon size except single-base substitution mutation by polyacrylamide gel electrophoresis (PAGE); suggesting that PAGE assay can be used for mutant detection, particularly in case where there was no enzyme restriction site in the spacer region between the target sites.2. Among the To plants of rice variety Kuiku131 transformed with Cas9/one-target-sgRNA-, Cas9/two-target-sgRNAs-and Cas9/three-target-sgRNAs expressing constructs (pC-ERF922S2, pC-ERP922S1S2 and pC-ERF922S1S2S3), the C-ERF922S2-, C-ERF922S1S2-and C-ERF922S1S2S3 targeting mutagenic frequencies of OsERF922 were 42.0%,70.0% and 90.0% and mutagenic frequencies of homozygous mutants were 14.3%,47.6% and 40.7%, respectively; indicting that the mutagenic frequencies increased when targeting more sites within one gene, and Cas9/two-targets-sgRNAs resulted in the highest mutagenic frequency in homozygotes. Furthermore, the C-ERF922S1S2S3 targeting mutagenic frequencies of OsERF92’2 were 90.0% and 92.5% in the To plants of rice varieties Kuiku131 and Jigeng 88 transformed with pC-ERF922S1S2S3, respectively; revealing that the mutagenic frequencies were no significant differences among different rice varieties by using the same Cas9/sgRNA.3. Among statistics of 325 Cas9/sgRNA-induced mutated events (mutated events not containing fragmental deletion between any two target sites and simultaneous fragmental deletion and insertion between any two target sites), more than half (55.1%,179/325) of the mutations were nucleotide deletions,39.4%(128/325) of the mutations were nucleotide insertions,4.3%(14/325) of the mutations were simultaneous nucleotide deletions and insertions, and 1.2%(4/325) of the mutations were single base substitution. In addition, mutations altered bases ranging from 1 bp to 200 bp, and mostly (52.3%,170/325) affected 1 base. As for the deletion mutations, the majority (69.8%,125/179) were short (<10 bp) deletions and the other 30.2%(54/325) were longer deletions ranging from 11 bp to 152 bp; as for the insertion mutations, almost all of them were single base insertion (96.1%,123/128), and the frequency of insert A (42.2%,54/128) or T (46.1%,59/128) far above insert G (1.5%, 2/128) or C (6.2%,8/128).4. Among analyzing transmission of CRISPR/Cas9-induced allele mutations from the To to the T1 and T2 generations, all allele mutations in 12 To and 5 T1 plants were stably transmitted to subsequent generations, and segregate to subsequent generations following Mendelian genetic law. In addition, the PCR assay failed to detect the T-DNA constructs in 18 out of 230 T1 plants derived from the 12 To mutant plants and the PCR assay also failed to detect the T-DNA constructs in 46 out of 147 T2 plants derived from the 5 T1 mutant plants in detetion of T-DNA-free plants; indicting that exogenous T-DNA fragment can be eliminated through genetic segregation in mutant plants.5. Resistance to M. oryzae were enhanced in all 6 T2 homozygous mutant rice lines compared with wild-type plants at both the seedling and tillering stages. Furthermore, there were no significant differences between any of the 6 T2 mutant lines and the wild-type with regard to the agronomic traits such as plant height, flag leaf length and width, number of productive panicles, panicle length, number of grains per panicle, seed setting percentage and thousand seed weight tested. These results indicate that resistance to M. oryzae were significantly improved and no significant change of agronomic traits in OsERF922 mutated rice lines.