The OsSPK1-OsRac1-RAI1 defense signaling pathway is shared by two distantly related NLR proteins in rice blast resistance.

Resistance (R) proteins are important components of plant innate immunity. Most known R proteins are nucleotide-binding site leucine-rich repeat (NLR) proteins. Although a number of signaling components downstream of NLRs have been identified, we lack a general understanding of the signaling pathways. Here, we used the interaction between rice (Oryza sativa) and Magnaporthe oryzae to study signaling of rice NLRs in response to blast infection. We found that in blast resistance mediated by the NLR PIRICULARIA ORYZAE RESISTANCE IN DIGU 3 (PID3), the guanine nucleotide exchange factor OsSPK1 works downstream of PID3. OsSPK1 activates the small GTPase OsRac1, which in turn transduces the signal to the transcription factor RAC IMMUNITY1 (RAI1). Further investigation revealed that the three signaling components also play important roles in disease resistance mediated by the distantly related NLR protein Pi9, suggesting that the OsSPK1-OsRac1-RAI1 signaling pathway could be conserved across rice NLR-induced blast resistance. In addition, we observed changes in RAI1 levels during blast infection, which led to identification of OsRPT2a, a subunit of the 19S regulatory particle of the 26S proteasome. OsRPT2a seemed to be responsible for RAI1 turnover in a 26S proteasome-dependent manner. Collectively, our results suggest a defense signaling route that might be common to NLR proteins in response to blast infection.

Importance of OsRac1 and RAI1 in signalling of nucleotide-binding site leucine-rich repeat protein-mediated resistance to rice blast disease.

Plants depend on Resistance (R) genes, most of which encode nucleotide-binding site leucine-rich repeat (NLR) proteins, for pathogen race-specific disease resistance. However, only a few immediate downstream targets of R proteins have been characterized, and the signalling pathways for R-protein-induced immunity are largely unknown. In rice (Oryza sativa), NLR proteins serve as important immune receptors in the response to rice blast disease caused by the fungus Magnaporthe oryzae. We used site-directed mutagenesis to create an autoactive form of the NLR protein PID3 that confers blast resistance and used transgenic rice to test the resulting immunity and gene expression changes. We identified OsRac1, a known GTPase, as a signalling molecule in PID3-mediated blast resistance, implicating OsRac1 as a possible common factor downstream of rice NLR proteins. We also identified RAI1, a transcriptional activator, as a PID3 interactor required for PID3-mediated blast resistance and showed that RAI1 expression is induced by PID3 via a process mediated by OsRac1. This study describes a new signalling pathway for NLR protein-mediated blast resistance and shows that OsRac1 and RAI1 act together to play a critical role in this process.

Identification of a G2-like transcription factor, OsPHL3, functions as a negative regulator of flowering in rice by co-expression and reverse genetic analysis.

BACKGROUND: Flowering time is a key trait for regional adaption and seed production in rice (Oryza sativa L.). Forward and reverse genetic studies have characterized a number of flowering-time genes. However, co-expression analysis has not been used to identify the flowering-time genes. RESULTS: We predicted a G2-like family transcription factor, OsPHL3, by co-expression networks analysis with photoperiodic flowering pathway genes. OsPHL3 contains a MYB-CC domain, and was localized in the nucleus with transcriptional activation potential. OsPHL3 was mainly expressed in the leaves and exhibited a circadian rhythmic expression pattern. Rice lines overexpressing OsPHL3 showed a delayed flowering time in the genetic background of TP309 under both long-day (Beijing) and short-day (Hainan) conditions. By contrast, the knockout rice lines of OsPHL3 by CRISPR/Cas9 technology promoted flowering time regardless of genetic backgrounds (i.e. Nipponbare and TP309) or day length. Further analysis indicated that OsPHL3 delayed flowering time by down-regulating the expression of Hd3a and RFT1 through promoting Hd1 under long-day conditions (LDs), or suppressing Ehd1/Hd1 under short-day conditions (SDs). CONCLUSIONS: Our results suggested that co-expression analysis is a useful strategy for identifying novel flowering-time genes in rice.

The E3 ligase OsPUB15 interacts with the receptor-like kinase PID2 and regulates plant cell death and innate immunity.

BACKGROUND: Rice blast disease is one of the most destructive diseases of rice worldwide. We previously cloned the rice blast resistance gene Pid2, which encodes a transmembrane receptor-like kinase containing an extracellular B-lectin domain and an intracellular serine/threonine kinase domain. However, little is known about Pid2-mediated signaling. RESULTS: Here we report the functional characterization of the U-box/ARM repeat protein OsPUB15 as one of the PID2-binding proteins. We found that OsPUB15 physically interacted with the kinase domain of PID2 (PID2K) in vitro and in vivo and the ARM repeat domain of OsPUB15 was essential for the interaction. In vitro biochemical assays indicated that PID2K possessed kinase activity and was able to phosphorylate OsPUB15. We also found that the phosphorylated form of OsPUB15 possessed E3 ligase activity. Expression pattern analyses revealed that OsPUB15 was constitutively expressed and its encoded protein OsPUB15 was localized in cytosol. Transgenic rice plants over-expressing OsPUB15 at early stage displayed cell death lesions spontaneously in association with a constitutive activation of plant basal defense responses, including excessive accumulation of hydrogen peroxide, up-regulated expression of pathogenesis-related genes and enhanced resistance to blast strains. We also observed that, along with plant growth, the cell death lesions kept spreading over the whole seedlings quickly resulting in a seedling lethal phenotype. CONCLUSIONS: These results reveal that the E3 ligase OsPUB15 interacts directly with the receptor-like kinase PID2 and regulates plant cell death and blast disease resistance.

Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice.

The rice blast resistance gene Pid3 encodes a nucleotide-binding-site leucine-rich repeat (NBS-LRR) protein. This gene was cloned from the rice 'Digu' (indica) by performing a genome-wide comparison of the NBS-LRR gene family between two genome-sequenced varieties, '9311' (indica) and 'Nipponbare' (japonica). In this study, we performed functional analysis of Pid3-A4, an ortholog of Pid3 revealed by allele mining in the common wild rice A4 (Oryza rufipogon). The predicted protein encoded by Pid3-A4 shares 99.03% sequence identity with Pid3, with only nine amino-acid substitutions. In wild rice plants, Pid3-A4 is constitutively expressed, and its expression is not induced by Magnaporthe oryzae isolate Zhong-10-8-14 infection. Importantly, in transgenic plants, Pid3-A4, as compared with Pid3, displays a distinct resistance spectrum to a set of M. oryzae isolates, including those that prevail in the rice fields of Sichuan Province. Therefore, Pid3-A4 should be quite useful for the breeding of rice blast resistance, especially in southwestern China.

A Rice NBS-ARC Gene Conferring Quantitative Resistance to Bacterial Blight Is Regulated by a Pathogen Effector-Inducible miRNA.

The bacterium Xanthomonas oryzae pv. Oryzae (Xoo) causes blight in rice worldwide, resulting in significant crop loss. However, no gene underlying a quantitative trait locus (QTL) for resistance against Xoo has been cloned yet. Here, we report the map-based cloning of a QTL, in which the NBS8R gene confers quantitative resistance to Xoo. NBS8R encodes an NB-ARC protein, which is involved in pathogen/microbe-associated molecular pattern-triggered immunity and whose expression is regulated by non-TAL effector XopQ-inducible Osa-miR1876 through DNA methylation. Sequence analysis of NBS8R in wild rice species and rice cultivars suggests that the Osa-miR1876 binding sites in the 5' UTR of NBS8R are inserted by chance and have undergone variations with Osa-miR1876 throughout evolution. The interaction between NBS8R and XopQ-inducible Osa-miR1876 is partially in keeping with the zigzag model, revealing that quantitative genes may also follow this model to control the innate immune response or basal disease resistance, and may prove valuable in utilizing the existing landraces that harbor the NBS8R gene but with no Osa-miR1876 binding site in rice breeding for bacterial blight resistance.

OsNPR3.3-dependent salicylic acid signaling is involved in recessive gene xa5-mediated immunity to rice bacterial blight.

Salicylic acid (SA) is a key natural component that mediates local and systemic resistance to pathogens in many dicotyledonous species. However, its function is controversial in disease resistance in rice plants. Here, we show that the SA signaling is involved in both pathogen-associated-molecular-patterns triggered immunity (PTI) and effector triggered immunity (ETI) to Xanthomonas oryzae pv. Oryzae (Xoo) mediated by the recessive gene xa5, in which OsNPR3.3 plays an important role through interacting with TGAL11. Rice plants containing homozygous xa5 gene respond positively to exogenous SA, and their endogenous SA levels are also especially induced upon infection by the Xoo strain, PXO86. Depletion of endogenous SA can significantly attenuate plant resistance to PXO86, even to 86∆HrpXG (mutant PXO86 with a damaged type III secretion system). These results indicated that SA plays an important role in disease resistance in rice plants, which can be clouded by high levels of endogenous SA and the use of particular rice varieties.

Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae.

Over recent decades, many pathogenicity genes of Magnaporthe oryzae have been identified but only a very limited number of genes have been identified that encode components of the conidiogenesis pathway. We report here a T-DNA insertional mutant that completely lost conidiation ability. Further investigation revealed that this mutant did not develop any conidiophore, and that the T-DNA was integrated into an annotated gene designated as conidiophore stalk-less1 or COS1. Complementation experiments suggested that COS1 may be a determinant of conidiation. Sequence analysis revealed that COS1 putatively encodes a 491-amino-acid zinc-finger protein and the protein was revealed localized to nucleus. Reverse-transcriptase polymerase chain reaction (RT-PCR)-based expression analysis indicated that two homologues of conidiophore-related genes were affected by the cos1 mutation, suggesting that Cos1 may function as a transcriptional regulator controlling genes responsible for conidiation. Inoculations of rice roots and wounded leaves with mycelia suggested that COS1 is not required for pathogenicity. Moreover, mutation of COS1 may aggravate infection of wounded leaves. Interestingly, different from the wild-type strain, mycelia of the cos1 mutant successfully infected host cells and caused visible symptoms on unwounded leaf blades and sheaths, indicating that Cos1 may have a role in some unknown mechanism of mycelial infection of M. oryzae.

Allelic variation of the rice blast resistance gene Pid3 in cultivated rice worldwide.

In this study, the re-sequencing data from 3,000 rice genomes project (3 K RGP) was used to analyze the allelic variation at the rice blast resistance (R) Pid3 locus. A total of 40 haplotypes were identified based on 71 nucleotide polymorphic sites among 2621 Pid3 homozygous alleles in the 3k genomes. Pid3 alleles in most japonica rice accessions were pseudogenes due to premature stop mutations, while those in most indica rice accessions were identical to the functional haplotype Hap_6, which had a similar resistance spectrum as the previously reported Pid3 gene. By sequencing and CAPS marker analyzing the Pid3 alleles in widespread cultivars in China, we verified that Hap_6 had been widely deployed in indica rice breeding of China. Thus, we suggest that the priority for utilization of the Pid3 locus in rice breeding should be on introducing the functional Pid3 alleles into japonica rice cultivars and the functional alleles of non-Hap_6 haplotypes into indica rice cultivars for increasing genetic diversity.

Endoplasmic reticulum membrane-bound MoSec62 is involved in the suppression of rice immunity and is essential for the pathogenicity of Magnaporthe oryzae.

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) constitutes the first line of plant inducible immunity. As an important step of plant colonization, phytopathogens have to suppress PTI, and secreted effectors are therefore co-evolved and deployed. In this study, we characterized the function of MoSec62 of Magnaporthe oryzae, the causal agent of the destructive rice blast. MoSec62 encodes a homologue of Sec62p, a yeast endoplasmic reticulum (ER) membrane transporter for precursors of secretory proteins. We showed that a T-DNA insertion into the promoter region of MoSec62, causing a disturbance to the up-regulation of MoSec62 expression during blast invasion, resulted in a complete loss of blast virulence of the mutant, M1575. Both 3,3'-diaminobenzidine (DAB) staining of the infected rice leaves and expression analysis revealed that the infectious attempt by the mutant led to strong defence responses of rice. Consistently, in transcriptomic analysis of rice leaves subject to blast inoculation, a battery of defence responses was found to be induced exclusively on M1575 challenge. For further exploration, we tested the pathogenicity on a highly susceptible rice variety and detected the accumulation of Slp1, a known PTI suppressor. Both results suggested that the mutant most likely failed to overcome rice PTI. In addition, we showed that MoSec62 was able to rescue the thermosensitivity of a yeast Δsec62, and the MoSec62-GFP fusion was co-localized to the ER membrane, both suggesting the conservation of Sec62 homologues. In conclusion, our data indicate that MoSec62, probably as an ER membrane transporter, plays an essential role in antagonizing rice defence at the early stages of blast invasion.

Expression Profiles, Characterization and Function of HbTCTP in Rubber Tree (Hevea brasiliensis).

As a highly conserved protein, the translationally controlled tumor protein (TCTP) carries out vital roles in various life processes. In rubber tree, two TCTP genes, HbTCTP and HbTCTP1, were cloned, but only HbTCTP1 was studied in details. In this study, cis-acting regulatory elements, expression patterns, subcellular localization, interacting proteins, and antioxidant activity of HbTCTP were systematically analyzed. Besides the common cis-acting regulatory elements, HbTCTP promoter also harbored various known cis-elements that respond to hormone/stresses. Being consistent with the aforementioned results, HbTCTP was regulated by drought, low temperature, high salt, ethylene (ET), wounding, H2O2, and methyl jasmonate (MeJA) treatments. HbTCTP was expressed throughout different tissues and developmental stages of leaves. In addition, HbTCTP was associated with tapping panel dryness (TPD). HbTCTP was localized in the membrane, cytoplasm and the nucleus, and interacted with four proteins rubber elongation factor (REF), 17.5 kDa heat shock family protein, annexin, and REF-like stress related protein 1. Being similar to HbTCTP1, HbTCTP also indicated antioxidant activity in metal-catalyzed oxidation (MCO) system. Our results are useful for further understanding the molecular characterization and expression profiles of HbTCTP, but also lay a solid foundation for elucidating the function of HbTCTP in rubber tree.

Excavation of Pid3 orthologs with differential resistance spectra to Magnaporthe oryzae in rice resource.

Twenty-six orthologs of the rice blast resistance gene Pid3 from cultivated varieties and wild rice accessions distributed in different areas were cloned by allele mining. Sequence analysis showed that while each of the orthologous genes from indica varieties and most wild accessions encodes a complete NBS-LRR protein, each of the proteins encoded by those from japonica varieties and few wild rice accessions presents a premature termination. Eleven of the 26 orthologs were selected for blast resistance testing by transforming into the blast susceptible rice variety TP309, respectively. Inoculation of 23 M. oryzae strains collected from diverse regions of China to the respective transgenic plants revealed that 6 Pid3 orthologs showed susceptible to all the tested strains, while the other 5 orthologs showed differential resistance spectra in a gradually spectrum-widen order as Pid3-W3, Pid3-W4, Pid3-I3, Pid3-W5 and Pid3-I1. Amino acid sequences alignment of these orthologs indicated that the sequence diversities between the blast resistance orthologs were mostly located in the LRR domain such as the substitutions of Q694H,D856H,Q896R,D899E etc. However, the differences between the resistance orthologs and the susceptible ones were mostly located in the NBS domain. The present experiments provide an example of that the ortholog evaluation of plant R genes could be an efficient way to expand the rice blast resistance and some other plant disease resistance as well for breeding.

Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae.

Agrobacterium tumefaciens-mediated transformation (ATMT) has been proven to be a powerful strategy for gene disruption in plants and fungi. Patterns associated with transferred DNA (T-DNA) integration in plants and yeast have been studied comprehensively, whereas no detailed analysis of T-DNA integration has been reported yet in filamentous fungi. Here, we reported the T-DNA insertion patterns in the genome of filamentous fungus Magnaporthe oryzae. Using ATMT, a T-DNA tagged population consisting of 6,179 transformants of M. oryzae was constructed. With thermal asymmetric interlaced-PCR (TAIL-PCR), 623 right border (RB) flanking sequences and 124 left border (LB) flanking sequences were generated. Analysis of these flanking sequences indicated a significant integration bias toward non-coding sequences, suggesting distribution of T-DNAs was not random. Comparing to T-DNA RB, LB was nicked inaccurately and truncated frequently during integration. Chromosomal rearrangements, such as deletion, inversion, and translocation, were associated with T-DNA integration in some transformants. Our data suggest that, comparing with plant cells, T-DNA integrates into this filamentous fungus with more precise and simpler patterns. Some phenotypic mutants were observed in our T-DNA tagged population, and these transformants will be very useful for functional genomics research of M. oryzae.