Bacillus amyloliquefaciens LM-1 Affects Multiple Cell Biological Processes in Magnaporthe oryzae to Suppress Rice Blast.

Magnaporthe oryzae, one of the most destructive rice pathogens, causes significant losses during the rice harvest every year. Bacillus amyloliquefaciens has been explored in many crops as a potential biocontrol agent. However, the mechanisms of B. amyloliquefaciens controled rice blast are not fully understood. Here, a biocontrol strain LM-1, isolated from a contaminated medium, was identified as B. amyloliquefaciens using morphological observation, physiological and biochemical tests, and 16S rDNA sequencing. LM-1 inhibited the growth and pathogenicity of M. oryzae and Bipolaris oryzae (Breda de Haan) Shoem. The mycelia of M. oryzae co-cultured with LM-1 were enlarged and broken by fluorescence microscopy using calcofluor white. LM-1 inhibited the mycelia of M. oryzae from producing conidia. Genes itu, srf, and fenB were detected in LM-1. Furthermore, the supernatant of LM-1 interfered with the appressorium formation of M. oryzae, blocked conidial cell death, and reduced autophagy degradation but did not affect the normal germination of rice seeds and seeding growth. Additionally, we observed hypersensitivity reactions, reactive oxygen species, and iron accumulation reduction in rice cells inoculated with supernatant. Our study reveals that LM-1 has a control effect on rice blast and affects cell wall integrity, sporulation, appressorium formation, cell death, and autophagy.

Identification and fine-mapping of Xo2, a novel rice bacterial leaf streak resistance gene.

KEY MESSAGE: A novel rice resistance gene, Xo2, influencing pathogenesis of the bacterial leaf streak disease, has been identified, and candidate genes for Xo2 in the fine mapping region have been shown to be involved in bacterial leaf streak resistance. Rice (Oryza sativa) bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola (Xoc), is one of the most serious rice bacterial diseases. The deployment of host resistance genes is an effective approach for controlling this disease. The cultivar BHADOIA 303 (X455) from Bangladesh is resistant to most of Chinese Xoc races. To identify and map the resistance gene(s) involved in Xoc resistance, we examined the association between phenotypic and genotypic variations in two F2 populations derived from crosses between X455/Jingang 30 and X455/Wushansimiao. The segregation ratios of the F2 progeny were consistent with the action of a single dominant resistance gene, which was designated as Xo2. Based on rice SNP chip (GSR40K) assays of X455, Jingang 30, and resistant and susceptible pools thereof, we mapped Xo2 to the region from 10 Mb to 12.5 Mb on chromosome 2. The target gene was further finely mapped between the markers RM12941 and D6-1 within an approximately 110-kb region. The de novo sequencing and gene annotation of X455 and Jingang 30 revealed nineteen predicted genes within the target region. RNA-seq and expression analysis showed that four candidate genes, including Osa002T0115800, encoding an NLR resistance protein, were distinctly upregulated. Differential sequence and synteny analysis between X455 and Jingang 30 suggested that Osa002T0115800 is likely the functional Xo2 gene. This study lays a foundation for marker-assisted selection resistance breeding against rice bacterial leaf streak and the further cloning of Xo2.

The OsOXO2, OsOXO3 and OsOXO4 Positively Regulate Panicle Blast Resistance in Rice.

BACKGROUND: Although panicle blast is more destructive to yield loss than leaf blast in rice, the cloned genes that function in panicle blast resistance are still very limited and the molecular mechanisms underlying panicle blast resistance remain largely unknown. RESULTS: In the present study, we have confirmed that the three Oxalate oxidase (OXO) genes, OsOXO2, OsOXO3 and OsOXO4 from a blast-resistant cultivar BC10 function in panicle blast resistance in rice. The expression of OsOXO2, OsOXO3 and OsOXO4 were induced by panicle blast inoculation. Subcellular localization analysis revealed that the three OXO proteins are all localized in the nucleus and cytoplasm. Simultaneous silencing of OsOXO2, OsOXO3 and OsOXO4 decreased rice resistance to panicle blast, whereas the OsOXO2, OsOXO3 and OsOXO4 overexpression rice plants individually showed enhanced panicle blast resistance. More H2O2 and higher expression levels of PR genes were observed in the overexpressing plants than in the control plants, while the silencing plants exhibited less H2O2 and lower expression levels of PR genes compared to the control plants. Moreover, phytohormone treatment and the phytohormone signaling related gene expression analysis showed that panicle blast resistance mediated by the three OXO genes was associated with the activation of JA and ABA signaling pathways but suppression of SA signaling pathway. CONCLUSION: OsOXO2, OsOXO3 and OsOXO4 positively regulate panicle blast resistance in rice. The OXO genes could modulate the accumulation of H2O2 and expression levels of PR gene in plants. Moreover, the OXO genes mediated panicle blast resistance could be regulated by ABA, SA and JA, and may be associated with the activation of JA and ABA signaling pathways but suppression of the SA signaling pathway.

Xa7, a Small Orphan Gene Harboring Promoter Trap for AvrXa7, Leads to the Durable Resistance to Xanthomonas oryzae Pv. oryzae.

BACKGROUND: The rice (Oryza sativa) gene Xa7 has been hypothesized to be a typical executor resistance gene against Xanthomonas oryzae pv. oryzae (Xoo), and has conferred durable resistance in the field for decades. Its identity and the molecular mechanisms underlying this resistance remain elusive. RESULTS: Here, we filled in gaps of genome in Xa7 mapping locus via BAC library construction, revealing the presence of a 100-kb non-collinear sequence in the line IRBB7 compared with Nipponbare reference genomes. Complementary transformation with sequentially overlapping subclones of the BACs demonstrated that Xa7 is an orphan gene, encoding a small novel protein distinct from any other resistance proteins reported. A 27-bp effector binding element (EBE) in the Xa7 promoter is essential for AvrXa7-inducing expression model. XA7 is anchored in the endoplasmic reticulum membrane and triggers programmed cell death in rice and tobacco (Nicotiana benthamiana). The Xa7 gene is absent in most cultivars, landraces, and wild rice accessions, but highly homologs of XA7 were identified in Leersia perrieri, the nearest outgroup of the genus Oryza. CONCLUSIONS: Xa7 acts as a trap to perceive AvrXa7 via EBEAvrXa7 in its promoter, leading to the initiation of resistant reaction. Since EBEAvrXa7 is ubiquitous in promoter of rice susceptible gene SWEET14, the elevated expression of which is conducive to the proliferation of Xoo, that lends a great benefit for the Xoo strains retaining AvrXa7. As a result, varieties harboring Xa7 would show more durable resistance in the field. Xa7 alleles analysis suggests that the discovery of new resistance genes could be extended beyond wild rice, to include wild grasses such as Leersia species.

Dynamics of the Rice Blast Fungal Population in the Field After Deployment of an Improved Rice Variety Containing Known Resistance Genes.

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most destructive diseases of rice worldwide. Management through the deployment of host resistance genes would be facilitated by understanding the dynamics of the pathogen's population in the field. Here, to investigate the mechanism underlying the breakdown of disease resistance, we conducted a six-year field experiment to monitor the evolution of M. oryzae populations in Qujiang from Guangdong. The new variety of Xin-Yin-Zhan (XYZ) carrying R genes Pi50 and Pib was developed using the susceptible elite variety, Ma-Ba-Yin-Zhan (MBYZ), as the recurrent line. Field trials of disease resistance assessment revealed that the disease indices of XYZ in 2012, 2013, 2016, and 2017 were 0.19, 0.39, 0.70, and 0.90, respectively, indicating that XYZ displayed a very rapid increase of disease severity in the field. To investigate the mechanism underlying the quick erosion of resistance of XYZ, we collected isolates from both XYZ and MBYZ for pathogenicity testing against six different isogenic lines. The isolates collected from XYZ showed a similar virulence spectrum across four different years whereas those from MBYZ showed increasing virulence to the Pi50 and Pib isogenic lines from 2012 to 2017. Molecular analysis of AvrPib in the isolates from MBYZ identified four different AvrPib haplotypes, i.e., AvrPib-AP1-1, AvrPib-AP1-2, avrPib-AP2, and avrPib-AP3, verified by sequencing. AvrPib-AP1-1 and AvrPib-AP1-2 are avirulent to Pib whereas avrPib-AP2 and avrPib-AP3 are virulent. Insertions of a Pot3 and an Mg-SINE were identified in avrPib-AP2 and avrPib-AP3, respectively. Two major lineages based on rep-PCR analysis were further deduced in the field population, implying that the field population is composed of genetically related isolates. Our data suggest that clonal propagation and quick dominance of virulent isolates against the previously resistant variety could be the major genetic events contributing to the loss of varietal resistance against rice blast in the field.

Identification of the novel bacterial blight resistance gene Xa46(t) by mapping and expression analysis of the rice mutant H120.

Rice bacterial leaf blight is caused by Xanthomonas oryzae pv. oryzae (Xoo) and produces substantial losses in rice yields. Resistance breeding is an effective method for controlling bacterial leaf blight disease. The mutant line H120 derived from the japonica line Lijiangxintuanheigu is resistant to all Chinese Xoo races. To identify and map the Xoo resistance gene(s) of H120, we examined the association between phenotypic and genotypic variations in two F2 populations derived from crosses between H120/CO39 and H120/IR24. The segregation ratios of F2 progeny consisted with the action of a single dominant resistance gene, which we named Xa46(t). Xa46(t) was mapped between the markers RM26981 and RM26984 within an approximately 65.34-kb region on chromosome 11. The 12 genes predicted within the target region included two candidate genes encoding the serine/threonine-protein kinase Doa (Loc_Os11g37540) and Calmodulin-2/3/5 (Loc_Os11g37550). Differential expression of H120 was analyzed by RNA-seq. Four genes in the Xa46(t) target region were differentially expressed after inoculation with Xoo. Mapping and expression data suggest that Loc_Os11g37540 allele is most likely to be Xa46(t). The sequence comparison of Xa23 allele between H120 and CBB23 indicated that the Xa46(t) gene is not identical to Xa23.

Synthesis of the cationic fluorescent probes for the detection of anionic surfactants by electrostatic self-assembly.

Anionic surfactants were widespread used in car cleaning agents, household detergents, agricultural and industrial processes, and considered as a major source of environmental pollutant. Therefore, it is necessary to develop a fast, simple, highly selective and sensitive probe for the detection of anionic surfactants. Here, we synthesized two aggregation induced emission (AIE)-active molecules 4,4',4″,4‴-(ethene-1,1,2,2-tetrayltetrakis(benzene-4,1-diyl))tetrakis (1-(4-bromobenzyl)pyridin-1-ium) bromide (TPE-Br) and 4,4',4″,4‴-(ethene-1,1,2,2-trayltetrakis(benzene-4,1-diyl))tetrakis(1-methylpyridin-1-ium)iodide (TPE-I), which were then applied as fluorescence probes for detecting sodium dodecyl sulfate (SDS) with high selectivity and sensitivity. In the presence of SDS, a multi-fold fluorescence emission intensity enhancement was observed in both two probes (TPE-Br and TPE-I) due to the electrostatic self-assembly of AIE molecular. The limits of detection are 71.5 and 120 nM for TPE-Br and TPE-I, respectively. This study may provide a new strategy for environmental monitoring by AIE-based fluorescent probe.

1,6-Elimination reaction induced detection of fluoride ions in vitro and in vivo based on a NIR fluorescent probe.

Near-Infrared "turn on" type fluorescent probes are attractive and promising in the fields of chemical sensing and bioimaging. Here, a new dicyanomethylene-4H-pyran derivative (DCM-Si) NIR fluorescent probe was designed and synthesized for specific lighting up F- in living cells and bodies. SiO bond was used as F- trigger, and the release of fluorophore (DCM-NH2) occurred after substituent reaction and subsequent 1,6-elimination. This NIR probe displayed high sensitivity and selectivity for the sensing of F-, and the detection limit was calculated to be as low as 157 nM. Moreover, the "off-on" fluorescent signal changes can be realized by adding F- in living cells and zebrafish embryos.

OsGF14e positively regulates panicle blast resistance in rice.

Though GF14e has been reported to negatively regulate bacterial blight and sheath blight resistance in rice, its effect on panicle blast, the most destructive disease in rice is still unknown. In the present study, we identified that GF14e was highly expressed in panicles and was induced in panicles infected by blast pathogen. Overexpression of GF14e enhances resistance to panicle blast whereas silencing GF14e results in increased susceptibility to panicle blast, suggesting that GF14e plays a positive role in quantitative panicle blast resistance in rice. Our results also demonstrate that GF14e is regulated by WRKY71 and GF14e-mediated panicle blast resistance is related to activation of SA-dependent pathway and suppression of JA-dependent pathway. The functional confirmation of GF14e in panicle blast resistance makes it to be a promising target in molecular rice breeding.

OsGF14b Positively Regulates Panicle Blast Resistance but Negatively Regulates Leaf Blast Resistance in Rice.

Although 14-3-3 proteins have been reported to be involved in responses to biotic stresses in plants, their functions in rice blast, the most destructive disease in rice, are largely unknown. Only GF14e has been confirmed to negatively regulate leaf blast. We report that GF14b is highly expressed in seedlings and panicles during blast infection. Rice plants overexpressing GF14b show enhanced resistance to panicle blast but are susceptible to leaf blast. In contrast, GF14b-silenced plants show increased susceptibility to panicle blast but enhanced resistance to leaf blast. Yeast one-hybrid assays demonstrate that WRKY71 binds to the promoter of GF14b and modulates its expression. Overexpression of GF14b induces expression of jasmonic acid (JA) synthesis-related genes but suppresses expression of salicylic acid (SA) synthesis-related genes. In contrast, suppressed GF14b expression causes decreased expression of JA synthesis-related genes but activation of SA synthesis-related genes. These results suggest that GF14b positively regulates panicle blast resistance but negatively regulates leaf blast resistance, and that GF14b-mediated disease resistance is associated with the JA- and SA-dependent pathway. The different functions for 14-3-3 proteins in leaf and panicle blast provide new evidence that leaf and panicle blast resistance are controlled by different mechanisms.

Functional divergence of duplicated genes results in a novel blast resistance gene Pi50 at the Pi2/9 locus.

KEY MESSAGE: We characterized a novel blast resistance gene Pi50 at the Pi2/9 locus; Pi50 is derived from functional divergence of duplicated genes. The unique features of Pi50 should facilitate its use in rice breeding and improve our understanding of the evolution of resistance specificities. Rice blast disease, caused by the fungal pathogen Magnaporthe oryzae, poses constant, major threats to stable rice production worldwide. The deployment of broad-spectrum resistance (R) genes provides the most effective and economical means for disease control. In this study, we characterize the broad-spectrum R gene Pi50 at the Pi2/9 locus, which is embedded within a tandem cluster of 12 genes encoding proteins with nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains. In contrast with other Pi2/9 locus, the Pi50 cluster contains four duplicated genes (Pi50_NBS4_1 to 4) with extremely high nucleotide sequence similarity. Moreover, these duplicated genes encode two kinds of proteins (Pi50_NBS4_1/2 and Pi50_NBS4_3/4) that differ by four amino acids. Complementation tests and resistance spectrum analyses revealed that Pi50_NBS4_1/2, not Pi50_NBS4_3/4, control the novel resistance specificity as observed in the Pi50 near isogenic line, NIL-e1. Pi50 shares greater than 96 % amino acid sequence identity with each of three other R proteins, i.e., Pi9, Piz-t, and Pi2, and has amino acid changes predominantly within the LRR region. The identification of Pi50 with its novel resistance specificity will facilitate the dissection of mechanisms behind the divergence and evolution of different resistance specificities at the Pi2/9 locus.