A natural gene drive system confers reproductive isolation in rice.

Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.

The rice RNase P protein subunit Rpp30 confers broad-spectrum resistance to fungal and bacterial pathogens.

RNase P functions either as a catalytic ribonucleoprotein (RNP) or as an RNA-free polypeptide to catalyse RNA processing, primarily tRNA 5' maturation. To the growing evidence of non-canonical roles for RNase P RNP subunits including regulation of chromatin structure and function, we add here a role for the rice RNase P Rpp30 in innate immunity. This protein (encoded by LOC_Os11g01074) was uncovered as the top hit in yeast two-hybrid assays performed with the rice histone deacetylase HDT701 as bait. We showed that HDT701 and OsRpp30 are localized to the rice nucleus, OsRpp30 expression increased post-infection by Pyricularia oryzae (syn. Magnaporthe oryzae), and OsRpp30 deacetylation coincided with HDT701 overexpression in vivo. Overexpression of OsRpp30 in transgenic rice increased expression of defence genes and generation of reactive oxygen species after pathogen-associated molecular pattern elicitor treatment, outcomes that culminated in resistance to a fungal (P. oryzae) and a bacterial (Xanthomonas oryzae pv. oryzae) pathogen. Knockout of OsRpp30 yielded the opposite phenotypes. Moreover, HA-tagged OsRpp30 co-purified with RNase P pre-tRNA cleavage activity. Interestingly, OsRpp30 is conserved in grass crops, including a near-identical C-terminal tail that is essential for HDT701 binding and defence regulation. Overall, our results suggest that OsRpp30 plays an important role in rice immune response to pathogens and provides a new approach to generate broad-spectrum disease-resistant rice cultivars.

Global Proteomic Analysis Reveals Widespread Lysine Succinylation in Rice Seedlings.

Lysine succinylation (Ksu) is a dynamic and reversible post-translational modification that plays an important role in many biological processes. Although recent research has analyzed Ksu plant proteomes, little is known about the scope and cellular distribution of Ksu in rice seedlings. Here, we report high-quality proteome-scale Ksu data for rice seedlings. A total of 710 Ksu sites in 346 proteins with diverse biological functions and subcellular localizations were identified in rice samples. About 54% of the sites were predicted to be localized in the chloroplast. Six putative succinylation motifs were detected. Comparative analysis with succinylation data revealed that arginine (R), located downstream of Ksu sites, is the most conserved amino acid surrounding the succinylated lysine. KEGG pathway category enrichment analysis indicated that carbon metabolism, tricarboxylic acid cycle (TCA) cycle, oxidative phosphorylation, photosynthesis, and glyoxylate and dicarboxylate metabolism pathways were significantly enriched. Additionally, we compared published Ksu data from rice embryos with our data from rice seedlings and found conserved Ksu sites between the two rice tissues. Our in-depth survey of Ksu in rice seedlings provides the foundation for further understanding the biological function of lysine-succinylated proteins in rice growth and development.

Transgenic Cotton Plants Expressing the HaHR3 Gene Conferred Enhanced Resistance to Helicoverpa armigera and Improved Cotton Yield.

RNA interference (RNAi) has been developed as an efficient technology. RNAi insect-resistant transgenic plants expressing double-stranded RNA (dsRNA) that is ingested into insects to silence target genes can affect the viability of these pests or even lead to their death. HaHR3, a molt-regulating transcription factor gene, was previously selected as a target expressed in bacteria and tobacco plants to control Helicoverpa armigera by RNAi technology. In this work, we selected the dsRNA-HaHR3 fragment to silence HaHR3 in cotton bollworm for plant mediated-RNAi research. A total of 19 transgenic cotton lines expressing HaHR3 were successfully cultivated, and seven generated lines were used to perform feeding bioassays. Transgenic cotton plants expressing dsHaHR3 were shown to induce high larval mortality and deformities of pupation and adult eclosion when used to feed the newly hatched larvae, and 3rd and 5th instar larvae of H. armigera. Moreover, HaHR3 transgenic cotton also demonstrated an improved cotton yield when compared with controls.

A proof-of-concept study in engineering synthetic protein for selective recognition of substrate-free polyubiquitin.

Similar to substrate-conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961-1969]. They designed a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnFCUBP domain, and a linkage-selective UBA domain, to specifically recognize unanchored Lys48-linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate-free Lys48-linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48-polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.

The ANIP1-OsWRKY62 module regulates both basal defense and Pi9-mediated immunity against Magnaporthe oryzae in rice.

During effector-triggered immunity (ETI) against the devastating rice blast pathogen Magnaporthe oryzae, Pi9 functions as an intracellular resistance protein sensing the pathogen-secreted effector AvrPi9 in rice. Importantly, the underlying recognition mechanism(s) between Pi9 and AvrPi9 remains elusive. In this study, we identified a rice ubiquitin-like domain-containing protein (UDP), AVRPI9-INTERACTING PROTEIN 1 (ANIP1), which is directly targeted by AvrPi9 and also binds to Pi9 in plants. Phenotypic analysis of anip1 mutants and plants overexpressing ANIP1 revealed that ANIP1 negatively modulates rice basal defense against M. oryzae. ANIP1 undergoes 26S proteasome-mediated degradation, which can be blocked by both AvrPi9 and Pi9. Moreover, ANIP1 physically associates with the rice WRKY transcription factor OsWRKY62, which also interacts with AvrPi9 and Pi9 in plants. In the absence of Pi9, ANIP1 negatively regulates OsWRKY62 abundance, which can be promoted by AvrPi9. Accordingly, knocking out of OsWRKY62 in a non-Pi9 background decreased immunity against M. oryzae. However, we also observed that OsWRKY62 plays negative roles in defense against a compatible M. oryzae strain in Pi9-harboring rice. Pi9 binds to ANIP1 and OsWRKY62 to form a complex, which may help to keep Pi9 in an inactive state and weaken rice immunity. Furthermore, using competitive binding assays, we showed that AvrPi9 promotes Pi9 dissociation from ANIP1, which could be an important step toward ETI activation. Taken together, our results reveal an immune strategy whereby a UDP-WRKY module, targeted by a fungal effector, modulates rice immunity in distinct ways in the presence or absence of the corresponding resistance protein.

Optimization of rice panicle architecture by specifically suppressing ligand-receptor pairs.

Rice panicle architecture determines the grain number per panicle and therefore impacts grain yield. The OsER1-OsMKKK10-OsMKK4-OsMPK6 pathway shapes panicle architecture by regulating cytokinin metabolism. However, the specific upstream ligands perceived by the OsER1 receptor are unknown. Here, we report that the EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE (EPFL) small secreted peptide family members OsEPFL6, OsEPFL7, OsEPFL8, and OsEPFL9 synergistically contribute to rice panicle morphogenesis by recognizing the OsER1 receptor and activating the mitogen-activated protein kinase cascade. Notably, OsEPFL6, OsEPFL7, OsEPFL8, and OsEPFL9 negatively regulate spikelet number per panicle, but OsEPFL8 also controls rice spikelet fertility. A osepfl6 osepfl7 osepfl9 triple mutant had significantly enhanced grain yield without affecting spikelet fertility, suggesting that specifically suppressing the OsEPFL6-OsER1, OsEPFL7-OsER1, and OsEPFL9-OsER1 ligand-receptor pairs can optimize rice panicle architecture. These findings provide a framework for fundamental understanding of the role of ligand-receptor signaling in rice panicle development and demonstrate a potential method to overcome the trade-off between spikelet number and fertility.

Resistosome and inflammasome: platforms mediating innate immunity.

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR) containing (NLR) proteins are intracellular immune receptors that sense pathogens or stress-associated signals in animals and plants. Direct or indirect binding of these stimuli to NLRs results in formation of higher-order large protein complexes termed inflammasomes in animals and resistosomes in plants to mediate immune signaling. Here we focus on plant NLRs and discuss the activation mechanism of the ZAR1 resistosome from Arabidopsis thaliana. We also outline the analogies and differences between the ZAR1 resistosome and the NLR inflammasomes, and discuss how the structural and biochemical information available on these two large types of protein complexes sheds light on signaling mechanisms of other plant NLRs.

New insights into the evolution of host specificity of three Penicillium species and the pathogenicity of P. Italicum involving the infection of Valencia orange (Citrus sinensis).

Blue and green molds, the common phenotypes of post-harvest diseases in fruits, are mainly caused by Penicillium fungal species, including P. italicum, P. digitatum, and P. expansum. We sequenced and assembled the genome of a P. italicum strain, which contains 31,034,623 bp with 361 scaffolds and 627 contigs. The mechanisms underlying the evolution of host specificity among the analyzed Penicillium species were associated with the expansion of protein families, genome restructuring, horizontal gene transfer, and positive selection pressure. A dual-transcriptome analysis following the infection of Valencia orange (Citrus sinensis) by P. italicum resulted in the annotation of 9,307 P. italicum genes and 24,591 Valencia orange genes. The pathogenicity of P. italicum may be due to the activation of effectors, including 51 small secreted cysteine-rich proteins, 110 carbohydrate-active enzymes, and 12 G protein-coupled receptors. Additionally, 211 metabolites related to the interactions between P. italicum and Valencia orange were identified by gas chromatography-time of flight mass spectrography, three of which were further confirmed by ultra-high performance liquid chromatography triple quadrupole mass spectrometry. A metabolomics analysis indicated that P. italicum pathogenicity is associated with the sphingolipid and salicylic acid signaling pathways. Moreover, a correlation analysis between the metabolite contents and gene expression levels suggested that P. italicum induces carbohydrate metabolism in Valencia orange fruits as part of its infection strategy. This study provides useful information regarding the genomic determinants that drive the evolution of host specificity in Penicillium species and clarifies the host-plant specificity during the infection of Valencia orange by P. italicum. IMPORTANCE: P. italicum GL_Gan1, a local strain in Guangzhou, China, was sequenced. Comparison of the genome of P. italicum GL_Gan1 with other pathogenic Penicillium species, P. digitatum and P. expansum, revealed that the expansion of protein families, genome restructuring, HGT, and positive selection pressure were related to the host range expansion of the analyzed Penicillium species. Moreover, gene gains or losses might be associated with the speciation of these Penicillium species. In addition, the molecular basis of host-plant specificity during the infection of Valencia orange (Citrus sinensis) by P. italicum was also elucidated by transcriptomic and metabolomics analysis. The data presented herein may be useful for further elucidating the molecular basis of the evolution of host specificity of Penicillium species and for illustrating the host-plant specificity during the infection of Valencia orange by P. italicum.

The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunity in Rice.

Programmed cell death (PCD) plays critical roles in plant immunity but must be regulated to prevent excessive damage. The E3 ubiquitin ligase SPL11 negatively regulates PCD and immunity in plants. We show that SPL11 cell-death suppressor 2 (SDS2), an S-domain receptor-like kinase, positively regulates PCD and immunity in rice by engaging and regulating SPL11 and related kinases controlling defense responses. An sds2 mutant shows reduced immune responses and enhanced susceptibility to the blast fungus Magnaporthe oryzae. Conversely, SDS2 over-expression induces constitutive PCD accompanied by elevated immune responses and enhanced resistance to M. oryzae. SDS2 interacts with and phosphorylates SPL11, which in turn ubiquitinates SDS2, leading to its degradation. In addition, SDS2 interacts with related receptor-like cytoplasmic kinases, OsRLCK118/176, that positively regulate immunity by phosphorylating the NADPH oxidase OsRbohB to stimulate ROS production. Thus, a plasma membrane-resident protein complex consisting of SDS2, SPL11, and OsRLCK118/176 controls PCD and immunity in rice.

Comparative Transcriptome Analysis of Penicillium citrinum Cultured with Different Carbon Sources Identifies Genes Involved in Citrinin Biosynthesis.

Citrinin is a toxic secondary metabolite of Penicillium citrinum and its contamination in many food items has been widely reported. However, research on the citrinin biosynthesis pathway and its regulation mechanism in P. citrinum is rarely reported. In this study, we investigated the effect of different carbon sources on citrinin production by P. citrinum and used transcriptome analysis to study the underlying molecular mechanism. Our results indicated that glucose, used as the sole carbon source, could significantly promote citrinin production by P. citrinum in Czapek's broth medium compared with sucrose. A total of 19,967 unigenes were annotated by BLAST in Nr, Nt, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Transcriptome comparison between P. citrinum cultured with sucrose and glucose revealed 1085 differentially expressed unigenes. Among them, 610 were upregulated while 475 were downregulated under glucose as compared to sucrose. KEGG pathway and Gene ontology (GO) analysis indicated that many metabolic processes (e.g., carbohydrate, secondary metabolism, fatty acid and amino acid metabolism) were affected, and potentially interesting genes that encoded putative components of signal transduction, stress response and transcription factor were identified. These genes obviously had important impacts on their regulation in citrinin biosynthesis, which provides a better understanding of the molecular mechanism of citrinin biosynthesis by P. citrinum.

Data for global lysine-acetylation analysis in rice (Oryza sativa).

Rice is one of the most important crops for human consumption and is a staple food for over half of the world׳s population (Yu et al., 2002) [1]. A systematic identification of the lysine acetylome was performed by our research (Xiong et al., 2016) [2]. Rice plant samples were collected from 5 weeks old seedlings (Oryza sativa, Nipponbare). After the trypsin digestion and immunoaffinity precipitation, LC-MS/MS approach was used to identify acetylated peptides. After the collected MS/MS data procession and GO annotation, the InterProScan was used to annotate protein domain. Subcellular localization of the identified acetylated proteins was predicted by WoLF PSORT. The KEGG pathway database was used to annotate identified acetylated protein interactions, reactions, and relations. The data, supplied in this article, are related to "A comprehensive catalog of the lysine-acetylation targets in rice (O. sativa) based on proteomic analyses" by Xiong et al. (2016) [2].

A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses.

Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in the gene transcription regulation. Here, we report high quality proteome-scale data for lysine-acetylation (Kac) sites and Kac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Kac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chloroplast. Seven putative acetylation motifs were detected. Phenylalanine, located in both the upstream and downstream of the Kac sites, is the most conserved amino acid surrounding the regions. In addition, protein interaction network analysis revealed that a variety of signaling pathways are modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways are significantly enriched. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development, and abiotic and biotic stress responses in rice and other plants. BIOLOGICAL SIGNIFICANCE: Rice is one of the most important crops consumption and is a model monocot for research. In this study, we combined a highly sensitive immune-affinity purification method (used pan anti-acetyl-lysine antibody conjugated agarose for immunoaffinity acetylated peptide enrichment) with high-resolution LC-MS/MS. In total, we identified 1337 Kac sites on 716 Kac proteins in rice cells. Bioinformatic analysis of the acetylome revealed that the acetylated proteins are involved in a variety of cellular functions and have diverse subcellular localizations. We also identified seven putative acetylation motifs in the acetylated proteins of rice. In addition, protein interaction network analysis revealed that a variety of signaling pathways were modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways were significantly enriched. To our knowledge, the number of Kac sites we identified was 23-times greater and the number of Kac proteins was 16-times greater than in a previous report. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development and responses to abiotic and biotic stresses in rice or other plants.

Silencing the HaHR3 gene by transgenic plant-mediated RNAi to disrupt Helicoverpa armigera development.

RNA interference (RNAi) caused by exogenous double-stranded RNA (dsRNA) has developed into a powerful technique in functional genomics, and to date it is widely used to down-regulate crucial physiology-related genes to control pest insects. A molt-regulating transcription factor gene, HaHR3, of cotton bollworm (Helicoverpa armigera) was selected as the target gene. Four different fragments covering the coding sequence (CDS) of HaHR3 were cloned into vector L4440 to express dsRNAs in Escherichia coli. The most effective silencing fragment was then cloned into a plant over-expression vector to express a hairpin RNA (hpRNA) in transgenic tobacco (Nicotiana tabacum). When H. armigera larvae were fed the E. coli or transgenic plants, the HaHR3 mRNA and protein levels dramatically decreased, resulting developmental deformity and larval lethality. The results demonstrate that both recombinant bacteria and transgenic plants could induce HaHR3 silence to disrupt H. armigera development, transgenic plant-mediated RNAi is emerging as a powerful approach for controlling insect pests.

Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60.

A novel antimicrobial peptide, with molecular mass of 1602.0469Da, produced by Brevibacillus laterosporus strain A60 was isolated and purified from the soil of mango plants. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on an HiTrap SP HP column, thin layer chromatography and High Performance Liquid Chromatography (HPLC) on C18 reversed-phase column. After the four isolation procedures, one peptide with antimicrobial activity was obtained and named BL-A60. The determination of the complete amino acid sequences of this peptide showed that it contains eleven amino acid residues, L-Y-K-L-V-K-V-V-L-N-M, and a choline connected to the N-terminal and a tenuazonic acid modified of the C-terminal. This peptide shows relatively low identification to other antimicrobial peptides from bacteria. Purified BL-A60 showed high pH and thermal stability and a strong inhibition of different stages of the life cycle of Phytophthora capsici, including mycelial growth, sporangia formation and cystospore germination, with EC(50) values of 7.89, 0.60 and 21.96 µg ml(-1), respectively.