Inducible Enrichment of Osa-miR1432 Confers Rice Bacterial Blight Resistance through Suppressing OsCaML2

MicroRNAs (miRNAs) handle immune response to pathogens by adjusting the function of target genes in plants. However, the experimentally documented miRNA/target modules implicated in the interplay between rice and Xanthomonas oryzae pv. oryzae (Xoo) are still in the early stages. Herein, the expression of osa-miR1432 was induced in resistant genotype IRBB5, but not susceptible genotype IR24, under Xoo strain PXO86 attack. Overexpressed osa-miR1432 heightened rice disease resistance to Xoo, indicated by enhancive enrichment of defense marker genes, raised reactive oxygen species (ROS) levels, repressed bacterial growth and shortened leaf lesion length, whilst the disruptive accumulation of osa-miR1432 accelerated rice susceptibility to Xoo infection. Noticeably, OsCaML2 (LOC_Os03g59770) was experimentally confirmed as a target gene of osa-miR1432, and the overexpressing OsCaML2 transgenic plants exhibited compromised resistance to Xoo infestation. Our results indicate that osa-miR1432 and OsCaML2 were differently responsive to Xoo invasion at the transcriptional level and fine-tune rice resistance to Xoo infection, which may be referable in resistance gene discovery and valuable in the pursuit of improving Xoo resistance in rice breeding.

Development of Markers for Identification and Maker-Assisted Breeding of Xa7 Gene in Rice (Oryza sativa L.)

Utilization of resistance (R) genes to breed resistant cultivars is one of the most effective and economical approach to control rice bacterial blight (BB). Xa7, a dominant, broad-spectrum and durable BB-resistant gene, is an ideal gene resource to improve the resistance of rice varieties to bacterial blight, and this well-known gene with important breeding value has been cloned in our recent study. The isolation of Xa7 will facilitate its application in rice breeding by molecular maker-assisted selection (MAS). In this study, based on the specific sequences in the promoter of Xa7, a functional marker, named as MX7, was developed, which can effectively distinguish the dominant BB-resistant Xa7, the recessive BB-susceptible xa7 and the null allele from different rice varieties. Since MX7 is a dominant marker, it can't tell homozygous from heterozygous, a co-dominant marker closely linked to Xa7, named as M6, was developed simultaneously. After verified by amplification in numerous rice varieties and sequence alignment in RICE 3K database, it is proved that marker M6 is co-segregated with the Xa7 locus. In addition, the effectiveness and accuracy of the two markers were further validated by two F2 populations. Finally, the designed makers were effectively applied in MAS breeding to improve the BB-resistance of a susceptible variety. This study not only provides reliable functional markers for the identification of Xa7 gene in different rice materials, but also will contribute to the application of Xa7 gene in marker-assisted selection to breed rice varieties with durable disease resistance.

TALE-triggered and iTALE-suppressed Xa1 resistance to bacterial blight is independent of OsTFIIAγ1 or OsTFIIAγ5 in rice

Xa1-mediated resistance to rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is triggered by transcription activator-like effectors (TALEs) and suppressed by interfering TALEs (iTALEs). TALEs interact with the rice transcription factor OsTFIIAγ1 or OsTFIIAγ5 (Xa5) to transcriptionally activate expression of target resistance and/or susceptibility genes. However, it is not clear whether OsTFIIAγ is involved in TALE-triggered and iTALE-suppressed Xa1 resistance. In this study, genome-edited mutations in OsTFIIAγ5 or OsTFIIAγ1 of Xa1-containing rice IRBB1 and Xa1-transgenic plants of xa5-containing rice IRBB5 did not impair the activation or suppression of Xa1 resistance. Correspondingly, the expression pattern of Xa1 in mutated OsTFIIAγ5 and OsTFIIAγ1 rice lines and IRBB1 rice was similar. In contrast, the expression of OsSWEET11 was repressed in mutated OsTFIIAγ5 and OsTFIIAγ1 rice lines. Biomolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) showed that both the TALE PthXo1 and iTALE Tal3a interacted with OsTFIIAγ1 and OsTFIIAγ5 in plant nuclei. These results indicate that TALE-triggered and iTALE-suppressed Xa1 resistance to BB is independent of OsTFIIAγ1 or OsTFIIAγ5 in rice and suggest that an unknown factor is potentially involved in the interaction of Xa1, TALEs and iTALEs in rice.

Enterobacter asburiae and Pantoea ananatis causing rice bacterial blight in China

Rice bacterial blight is a devastating bacterial disease threatening rice yield all over the world and Xanthomonas oryzae pv. oryzae (Xoo) is traditionally believed as the pathogen. In recent years, we have received diseased rice samples with symptoms of blight leaves from Sichuan and Guangdong Provinces, China. Pathogen isolation and classification identified two different enterobacteria as the causal agents, namely as Enterobacter asburiae and Pantoea ananatis. Among them, E. asburiae was isolated from samples of both Provinces, and P. ananatis was only isolated from the Sichuan samples. Different from rice foot rot pathogen Dickeya zeae EC1 and rice bacterial blight pathogen Xoo PXO99A, strains SC1, RG1 and SC7 produced rare cell wall degrading enzymes (CWDEs) but more extrapolysaccharides (EPS). E. asburiae strains SC1 and RG1 produced bacteriostatic substances while P. ananatis strain SC7 produced none. Pathogenicity tests indicated that all of them infected monocotyledonous rice and banana seedlings, but not dicotyledonous potato, radish or cabbage. Moreover, strain RG1 was most virulent, while strains SC1 and SC7 were similar virulent on rice leaves, even though strain SC1 propagated significantly faster in rice leaf tissues than strain SC7. This study firstly discovered E. asburiae as a new pathogen of rice bacterial blight, and in some cases, P. ananatis could be a companion pathogen. Analysis on production of virulence factors suggested that both pathogens probably employ a different mechanism to infect hosts other than using cell wall degrading enzymes to break through host cell walls.

Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.