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Odorant-binding proteins (OBPs) are crucial in insect olfaction. The most abundant expressed OBP of citrus psyllids, DcitOBP9 encodes 148 amino acids. DcitOBP9 lacks a transmembrane structure and possesses a 17-amino acid signal peptide at the N-terminus. Characterized by the six conserved cysteine sites, DcitOBP9 is classified as the Classical-OBP family. RT-qPCR experiments revealed ubiquitous expression of DcitOBP9 across all developmental stages of the citrus psyllid, with predominant expression in adults antennae. Fluorescence competitive binding assays demonstrated DcitOBP9's strong affinity for ocimene, linalool, dodecanoic acid, and citral, and moderate affinity for dimethyl trisulfide. Additionally, it binds to myrcia, (-)-trans-caryophyllene, (±)-Citronellal, nonanal, and (+)-α-pinene. Among them, ocimene, linalool, and dodecanoic acid were dynamically bound to DcitOBP9, while citral was statically bound to DcitOBP9. Molecular docking simulations with the top five ligands indicated that amino acid residues V92, S72, P128, L91, L75, and A76 are pivotal in the interaction between DcitOBP9 and these odorants. These findings suggest DcitOBP9's involvement in the citrus psyllid's host plant recognition and selection behaviors, thereby laying a foundation for elucidating the potential physiological and biological functions of DcitOBP9 and developing attractants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The root-knot nematode Meloidogyne graminicola secretes effectors into rice tissues to modulate host immunity. Here, we characterised MgCRT1, a calreticulin protein of M. graminicola, and identified its target in the plant. In situ hybridisation showed MgCRT1 mRNA accumulating in the subventral oesophageal gland in J2 nematodes. Immunolocalization indicated MgCRT1 localises in the giant cells during parasitism. Host-induced gene silencing of MgCRT1 reduced the infection ability of M. graminicola, while over-expressing MgCRT1 enhanced rice susceptibility to M. graminicola. A yeast two-hybrid approach identified the calmodulin-like protein OsCML31 as an interactor of MgCRT1. OsCML31 interacts with the high mobility group protein OsHMGB1 which is a conserved DNA binding protein. Knockout of OsCML31 or overexpression of OsHMGB1 in rice results in enhanced susceptibility to M. graminicola. In contrast, overexpression of OsCML31 or knockout of OsHMGB1 in rice decreases susceptibility to M. graminicola. The GST-pulldown and luciferase complementation imaging assay showed that MgCRT1 decreases the interaction of OsCML31 and OsHMGB1 in a competitive manner. In conclusion, when M. graminicola infects rice and secretes MgCRT1 into rice, MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1, resulting in the release of OsHMGB1 to enhance rice susceptibility., (© 2024 John Wiley & Sons Ltd.)

Wolbachia , a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus ( C Las) that causes citrus greening disease, is naturally infected with Wolbachia ( w Di). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 w Di genomes to gain a comprehensive understanding of w Di. Based on average nucleotide identity (ANI) analysis, we classified the w Di strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed w Di genomes. The variation analysis between Asia and North America w Di showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of w Di contributes to our understanding of the geographical population of w Di, the origin of hosts of D. citri , and the interaction between w Di and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

Rice blast, caused by Magnaporthe oryzae is one of the most destructive diseases of rice ( Oryza sativa L.) in most rice-cultivated areas worldwide. Mowanggu (MWG) is a traditional landrace rice variety in Yunnan with broad-spectrum and durable blast resistance against rice blast fungus. However, the underlying disease-resistance mechanisms remain unknown. An integrative transcriptomic, proteomic, and phosphoproteomic analysis of MWG was performed after inoculation with M. oryzae in this study. The transcriptomic and proteomic results revealed that MWG was moderately correlated at the transcriptional and protein levels. Differentially expressed genes and proteins were up-regulated and significantly enriched in protein phosphorylation, peroxisome, plant-pathogen interactions, phenylpropanoid metabolism and phenylalanine biosynthesis pathways. The phosphoproteomic profile and phosphorylated-protein-interaction network revealed that the altered phosphoproteins were primarily associated with reactive oxygen species (ROS), glycolysis, MAPK signaling pathways, and amino acid biosynthesis. In addition, a series of physiological and biochemical parameters, including ROS, soluble sugars, soluble protein and callus accumulation and defense-related enzyme activities, were used to validate the possible blast resistance mechanisms of MWG. The integrative transcriptomic, proteomic, and phosphoproteomic analysis revealed the different expression patterns at the molecular level of the durably resistant rice cultivar MWG after inoculation with M. oryzae , which provides insight into the molecular mechanisms of rice blast resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Peng, Wang, Zeng, Li, Song, Liu, Wang and Dai.)

Phytophthora sojae causes Phytophthora root and stem rot disease of soybean (Glycine max), leading to huge annual yield loss worldwide, but resistance to Phytophthora sojae (Rps) genes remains elusive. Soybean cultivar "Yudou 29" is resistant to P. sojae strain PsMC1, and this study aimed to clone, identify, and characterize the Rps gene in Yudou 29 (RpsYD29) and clarify its functional mechanism. We map-based cloned RpsYD29 (ZINC FINGER PROTEIN03, GmZFP03) using the families of a cross between Yudou 29 and a P. sojae-susceptible soybean cultivar "Jikedou 2". P. sojae resistance of GmZFP03 was functionally validated by stable soybean genetic transformation and allele-phenotype association analysis. GmZFP03 was identified as a C2H2-type zinc finger protein transcription factor, showing 4 amino acid residue polymorphisms (V79F, G122-, G123-, and D125V) and remarkably different expression patterns between resistant and susceptible soybeans. Notably boosted activity and gene expression of superoxide dismutase (SOD) in resistant-type GmZFP03-expressed transgenic soybean, substantial enhancement of P. sojae resistance of wild-type soybean by exogenous SOD treatment, and GmZFP03 binding to and activation of 2 SOD1 (Glyma.03g242900 and Glyma.19g240400) promoters demonstrated the involvement of SOD1s in GmZFP03-mediated resistance to P. sojae strain PsMC1. Thus, this study cloned the soybean P. sojae-resistant GmZFP03, the product of which specifically targets 2 SOD1 promoters. GmZFP03 can be directly used for precise P. sojae-resistance soybean breeding., Competing Interests: Conflict of interest statement. None declared ., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In China, citrus Huanglongbing (HLB) disease is caused by the Candidatus Liberibacter asiaticus bacterium, which is carried by the Asian citrus psyllid Diaphorina citri Kuwayama. It was hypothesized that the epidemic of the HLB may related with the rate of bacterium presence in the insect vector and bacterium content in plant tissues, as well as the phyllosphere microbe communities changes. This study systematically analyzed the presence or absence of Ca. L. asiaticus in citrus tree leaves and in the insect vector D. citri over a 6-year period using real-time PCR. In addition, changes in the number of bacteria carried by D. citri over 12 months were quantified, as well as the relationship between the proportion of D. citri carrying Ca. L. asiaticus and the proportion of plants infected with Ca. L. asiaticus were analyzed. Results showed that the proportion of D. citri carrying bacteria was stable and relatively low from January to September. The bacteria in citrus leaves relatively low in spring and summer, then peaked in December. The proportion of D. citri carrying bacteria gradually declined from 2014 to 2019. The proportion of D. citri carrying Ca. L. asiaticus showed a significant positive correlation with the proportion of diseased citrus. The phyllosphere bacterial and fungal communities on the healthy citrus leaf were significantly different with the disease leaf in April and December. Pathogenic invasions change the citrus phyllosphere microbial community structure. It could be summarized that citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hu, Meng, Yao, Wang, Tang, Wang, Dai, Zhao, Zhang, Fan, Luo, Xiang and Zhang.)

Rhizosphere-associated microbes have important implications for plant health, but knowledge of the association between the pathological conditions of soil-borne virus-infected wheat and soil microbial communities, especially changes in fungal communities, remains limited. We investigated the succession of fungal communities from bulk soil to wheat rhizosphere soil in both infected and healthy plants using amplicon sequencing methods, and assessed their potential role in plant health. The results showed that the diversity of fungi in wheat rhizosphere and bulk soils significantly differed post wheat yellow mosaic virus disease onset. The structure differences in fungal community at the two wheat health states or two compartment niches were evident, soil physicochemical properties (i.e., NH 4 + ) contribute to differences in fungal community structure and alpha diversity. Comparison analysis showed Mortierellomycetes and Dothideomycetes as dominant communities in healthy wheat soils at class level. The genus Pyronemataceae and Solicoccozyma were significantly are significantly enriched in rhizosphere soil of diseased plant, the genus Cystofilobasidium , Cladosporium , Mortierella , and Stephanonectria are significantly enriched in bulk soil of healthy plant. Co-occurrence network analysis showed that the fungi in healthy wheat soil has higher mutual benefit and connectivity compared with diseased wheat. The results of this study demonstrated that the occurrence of wheat yellow mosaic virus diseases altered both fungal community diversity and composition, and that NH 4 + is the most important soil physicochemical factor influencing fungal diversity and community composition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Hu, Cai, Wu, Zhang, Wei, Zhang, Hu, Liu, Lei, Ge, Dai, Yang and Chen.)

Background: Biotechnologists seeking to develop marker-free transgenic plants have established co-transformation methods. For co-transformation using mixed Agrobacterium strains, the mix ratio of Agrobacterium strains and selection scheme may influence co-transformation frequency. This study used fluorescent GFP and RFP markers to compose different selection schemes for observation of the selective dynamics of transformed rice cells and to investigate the factors affecting co-transformation efficiency., Methods and Results: We utilized GFP and RFP markers in co-transformation and tested the combinations of an antibiotic-selectable vector (pGFP-HPT) and a single RFP vector (pRFP) and of two antibiotic-selectable vectors (pGFP-HPT and pRFP-HPT) in rice. The pGFP-HPT/pRFP combination resulted in 70.9% to 81.2% of co-transformation frequencies while lower frequencies (56.6% on average) were obtained with the pGFP-HPT/pRFP-HPT combination. Based on GFP/RFP segregation patterns, 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny, which simulated the selection process of marker-free transgenic plants that carry an actual gene of interest. Transgene expression levels in the rice lines varied as revealed by RT-PCR, and tandem-linked T-DNAs were detected in co-transformants, suggesting that transgene expression might be affected by duplicated T-DNA structures., Conclusion: Co-transformation via mixed Agrobacterium strains is feasible, and approximately 55% of the pGFP-HPT/pRFP co-transformants contained unlinked T-DNAs and segregated single RFP progeny. The pGFP-HPT/pRFP and the pGFP-HPT/pRFP-HPT vector combinations showed distinctive selective dynamics of transformed rice cells, suggesting that co-transformation efficiency depends on both vector system and selection scheme., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Edeines, a group of cationic antimicrobial peptides produced by the soil bacterium Brevibacillus, have broad biological effects, such as antimicrobial, anticancer and immunosuppressive activities. However, the yield of edeines in wild-type (WT) Brevibacillus is extremely low, and chemical synthesis of edeines is a time-consuming process. Genetic engineering has proven to be an effective approach to produce antibiotics with high yield. In this study, the edeine biosynthetic gene cluster (ede BGC), which is involved in edeine production, was identified and characterized in Brevibacillus brevis X23. To improve edeine production in B. brevis X23, the ede BGC promoter was replaced with six different promoters, P mwp , P spc , P xylA , P shuttle-09 , P grac or P 43 , through double-crossover homologous recombination. The new promoters significantly increased the expression of the ede BGC as well as edeine production by 2.9 ± 0.4 to 20.5 ± 1.2-fold and 3.6 ± 0.1to 8.7 ± 0.7-fold respectively. The highest yield of edeines (83.6 mg l -1 ) was obtained in B. brevis X23 with the P mwp promoter. This study provides a practical approach for producing high yields of edeines in B. brevis., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Three-amino-acid-loop-extension (TALE) transcription factors comprise one of the largest gene families in plants, in which they contribute to regulation of a wide variety of biological processes, including plant growth and development, as well as governing stress responses. Although sweet orange ( Citrus sinensis ) is among the most commercially important fruit crops cultivated worldwide, there have been relatively few functional studies on TALE genes in this species. In this study, we investigated 18 CsTALE gene family members with respect to their phylogeny, physicochemical properties, conserved motif/domain sequences, gene structures, chromosomal location, cis -acting regulatory elements, and protein-protein interactions (PPIs). These CsTALE genes were classified into two subfamilies based on sequence homology and phylogenetic analyses, and the classification was equally strongly supported by the highly conserved gene structures and motif/domain compositions. CsTALEs were found to be unevenly distributed on the chromosomes, and duplication analysis revealed that segmental duplication and purifying selection have been major driving force in the evolution of these genes. Expression profile analysis indicated that CsTALE genes exhibit a discernible spatial expression pattern in different tissues and differing expression patterns in response to different biotic/abiotic stresses. Of the 18 CsTALE genes examined, 10 were found to be responsive to high temperature, four to low temperature, eight to salt, and four to wounding. Moreover, the expression of CsTALE3/8/12/16 was induced in response to infection with the fungal pathogen Diaporthe citri and bacterial pathogen Candidatus Liberibacter asiaticus, whereas the expression of CsTALE15/17 was strongly suppressed. The transcriptional activity of Cs TALE proteins was also verified in yeast, with yeast two-hybrid assays indicating that CsTALE3/CsTALE8, CsTALE3/CsTALE11, CsTALE10/CsTALE12, CsTALE14/CsTALE8, CsTALE14/CsTALE11 can form respective heterodimers. The findings of this study could lay the foundations for elucidating the biological functions of the TALE family genes in sweet orange and contribute to the breeding of stress-tolerant plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peng, Yang, Xu, Peng, Dai, Dai, Wang, Yi, Wang, Li and Song.)

The soil microbial community plays a vital role in the biogeochemical cycles of bioelements and maintaining healthy soil conditions in agricultural ecosystems. However, how the soil microbial community responds to mitigation measures for continuous cropping obstacles remains largely unknown. Here we examined the impact of quicklime (QL), chemical fungicide (CF), inoculation with earthworm (IE), and a biocontrol agent (BA) on the soil microbial community structure, and the effects toward alleviating crop yield decline in lily. High-throughput sequencing of the 16S rRNA gene from the lily rhizosphere after 3 years of continuous cropping was performed using the Illumina MiSeq platform. The results showed that Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Gemmatimonadetes were the dominant bacterial phyla, with a total relative abundance of 86.15-91.59%. On the other hand, Betaproteobacteriales, Rhizobiales, Myxococcales, Gemmatimonadales, Xanthomonadales, and Micropepsales were the dominant orders with a relative abundance of 28.23-37.89%. The hydrogen ion concentration (pH) and available phosphorus (AP) were the key factors affecting the structure and diversity of the bacterial community. The yield of continuous cropping lily with using similar treatments decreased yearly for the leaf blight, but that of IE was significantly (p < 0.05) higher than with the other treatments in the same year, which were 17.9%, 18.54%, and 15.69% higher than that of blank control (CK) over 3 years. In addition, IE significantly (p < 0.05) increased organic matter (OM), available nitrogen (AN), AP, and available potassium (AK) content in the lily rhizosphere soil, optimized the structure and diversity of the rhizosphere bacterial community, and increased the abundance of several beneficial bacterial taxa, including Rhizobiales, Myxococcales, Streptomycetales and Pseudomonadales. Therefore, enriching the number of earthworms in fields could effectively optimize the bacterial community structure of the lily rhizosphere soil, promote the circulation and release in soil nutrients and consequently alleviate the loss of continuous cropping lily yield., (© 2021. The Author(s).)

Rice blast caused by Magnaporthe oryzae is one of the most important diseases that seriously threaten rice production. Brachypodium distachyon is a grass species closely related to grain crops, such as rice, barley, and wheat, and has become a new model plant of Gramineae. In this study, 15 small RNA samples were sequenced to examine the dynamic changes in microRNA (miRNA) expression in B. distachyon infected by M. oryzae at 0, 24, and 48 h after inoculation. We identified 432 conserved miRNAs and 288 predicted candidate miRNAs in B. distachyon . Additionally, there were 7 and 19 differentially expressed miRNAs at 24 and 48 h post-inoculation, respectively. Furthermore, using degradome sequencing, we identified 2,126 genes as targets for 308 miRNAs; using quantitative real-time PCR (qRT-PCR), we validated five miRNA/target regulatory units involved in B. distachyon-M. oryzae interactions. Moreover, using co-transformation technology, we demonstrated that BdNAC21 was negatively regulated by miR164c. This study provides a new approach for identifying resistance genes in B. distachyon by mining the miRNA regulatory network of host-pathogen interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Peng, Song, Li, Yan, Huang, Yang, Duan, Dai and Wang.)

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&#95;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., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

SBP-box is an important plant-specific transcription factor family and is involved in diverse biological processes. Here, we identified a total of 15 SBP-BOX genes in the important fruit crop sweet orange ( Citrus sinensis ) and characterized their gene structures, conserved domain and motif, chromosomal location, and cis -acting regulatory elements. SBP genes were classified into four subfamilies based on the amino acid sequence homology, and the classification is equally strongly supported by the gene and protein structures. Our analysis revealed that segmental duplication events were the main driving force in the evolution of CsSBP genes, and gene pairs might undergo extensive purifying selection. Further synteny analysis of the SBP members among sweet orange and other plant species provides valuable information for clarifying the CsSBP family evolutionary relationship. According to publicly available RNA-seq data and qRT-PCR analysis from various sweet orange tissues, CsSBP genes may be expressed in different tissues and developmental stages. Gene expression analysis showed variable expression profiles of CsSBP genes under various abiotic stresses, such as high and low-temperature, salt, and wound treatments, demonstrating the potential role of SBP members in sweet orange response to abiotic stress. Noticeably, all CsSBP genes were also downregulated in sweet orange upon the infection of an important fungal pathogen Diaporthe citri . Our results provide valuable information for exploring the role of SBP-Box in sweet orange.

P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G 2 A 6 slippery site in the genus Potyvirus . However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae . Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus ) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus ) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G 2 A 6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G 2 A 6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae . To the best of our knowledge, this is the first experimental evidence for the expression mechanism of P3N-PIPO in the genus Bymovirus , the only genus comprising bipartite virus species in the family Potyviridae .

Recent studies have shown that a large number of long noncoding RNAs (lncRNAs) can regulate various biological processes in animals and plants. Although lncRNAs have been identified in many plants, they have not been reported in the model plant Nicotiana benthamiana . Particularly, the role of lncRNAs in plant virus infection remains unknown. In this study, we identified lncRNAs in N. benthamiana response to Chinese wheat mosaic virus (CWMV) infection by RNA sequencing. A total of 1175 lncRNAs, including 65 differentially expressed lncRNAs, were identified during CWMV infection. We then analyzed the functions of some of these differentially expressed lncRNAs. Interestingly, one differentially expressed lncRNA, XLOC&#95;006393, was found to participate in CWMV infection as a precursor to microRNAs in N. benthamiana . These results suggest that lncRNAs play an important role in the regulatory network of N. benthamiana in response to CWMV infection.

The GATA proteins, functioning as transcription factors (TFs), are involved in multiple plant physiological and biochemical processes. In this study, 28 GATA TFs of Brachypodium distachyon (BdGATA) were systematically characterized via whole-genome analysis. BdGATA genes unevenly distribute on five chromosomes of B. distachyon and undergo purifying selection during the evolution process. The putative cis -acting regulatory elements and gene interaction network of BdGATA were found to be associated with hormones and defense responses. Noticeably, the expression profiles measured by quantitative real-time PCR indicated that BdGATA genes were sensitive to methyl jasmonate (MeJA) and salicylic acid (SA) treatment, and 10 of them responded to invasion of the fungal pathogen Magnaporthe oryzae , which causes rice blast disease. Genome-wide characterization, evolution, and expression profile analysis of BdGATA genes can open new avenues for uncovering the functions of the GATA genes family in plants and further improve the knowledge of cellular signaling in plant defense.

Myzus persicae (Sulzer), commonly known as the green peach aphid, is a notorious pest that causes substantial losses to a range of crops and can transmit several plant viruses, including potato virus Y (PVY). Chemical insecticides provide only partial control of this pest and their use is not environmentally sustainable. In recent years, many genes related to growth, development, and reproduction have been used as targets for pest control. These include Gustavus ( Gus ), a highly conserved gene that has been reported to play an essential part in the genesis of germline cells and, hence, in fecundity in the model insect Drosophila melanogaster . We hypothesized that the Gustavus ( Gus ) gene was a potential target that could be used to regulate the M. persicae population. In this study, we report the first investigation of an ortholog of Gus in M. persicae , designated MpGus , and describe its role in the fecundity of this insect. First, we identified the MpGus mRNA sequence in the M. persicae transcriptome database, verified its identity with reverse transcription-polymerase chain reaction (RT-PCR), and then evaluated the transcription levels of MpGus in M. persicae nymphs of different instars and tissues with real-time quantitative PCR (RT-qPCR). To investigate its role in regulating the fecundity of M. persicae , we used RNA interference (RNAi) to silence the expression of MpGus in adult insects; this resulted in a significant reduction in the number of embryos (50.6%, P < 0.01) and newborn nymphs (55.7%, P < 0.01) in the treated aphids compared with controls. Interestingly, MpGus was also significantly downregulated in aphids fed on tobacco plants that had been pre-infected with PVY N , concomitant with a significant reduction (34.1%, P < 0.01) in M. persicae fecundity. Collectively, these data highlight the important role of MpGus in regulating fecundity in M. persicae and indicate that MpGus is a promising RNAi target gene for control of this pest species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gao, Ren, Peng, Wang, Shi, Zheng, Zhang, Zhu, Liu, Dai and Zhang.)

Histone acetylation is a dynamic modification process co-regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HDACs play vital roles in abiotic or biotic stress responses, their members in Triticum aestivum and their response to plant viruses remain unknown. Here, we identified and characterized 49 T. aestivum HDACs (TaHDACs) at the whole-genome level. Based on phylogenetic analyses, TaHDACs could be divided into 5 clades, and their protein spatial structure was integral and conserved. Chromosomal location and synteny analyses showed that TaHDACs were widely distributed on wheat chromosomes, and gene duplication has accelerated the TaHDAC gene family evolution. The cis-acting element analysis indicated that TaHDACs were involved in hormone response, light response, abiotic stress, growth, and development. Heatmaps analysis of RNA-sequencing data showed that TaHDAC genes were involved in biotic or abiotic stress response. Selected TaHDACs were differentially expressed in diverse tissues or under varying temperature conditions. All selected TaHDACs were significantly upregulated following infection with the barley stripe mosaic virus (BSMV), Chinese wheat mosaic virus (CWMV), and wheat yellow mosaic virus (WYMV), suggesting their involvement in response to viral infections. Furthermore, TaSRT1 -silenced contributed to increasing wheat resistance against CWMV infection. In summary, these findings could help deepen the understanding of the structure and characteristics of the HDAC gene family in wheat and lay the foundation for exploring the function of TaHDACs in plants resistant to viral infections.

The Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), is a notorious Rutaceae plant pest. Frequent and extensive use of pesticides has resulted in severe insecticide resistance in ACP populations. Fully understanding the mechanism of ACP resistance to pesticides is vital for us to control or delay the development of resistance. Therefore, we compared the difference in resistance to imidacloprid and thiamethoxam between Hunan (Yongzhou, Chenzhou) and Guangdong (Guangzhou) ACP populations and analyzed the correlations between the resistance level and genes and symbiotic fungi. The results showed that the resistance of the Guangdong ACP population to imidacloprid and thiamethoxam was lower than that of Hunan ACP population, and the relative expression of genes associated with P450 mono-oxygenase and acetylcholinesterase was significantly lower in the Guangdong ACP population than in Hunan ACP population. The differences of mean relative abundances of four symbiotic bacteria among three populations were marginally significant; however, the mean relative abundance of 16 fungi among three populations was significantly different, and positive linear correlations were observed between the resistance level and two fungi ( Aspergillus niger and Aureobasidium pullulans ) and two genes ( CYP4C70 and CYP4DB1 ). Negative correlations were only observed between the resistance level and two fungi ( Golubevia pallescens and Acremonium sclerotigenum ). Moreover, four fungi were unique to the Chenzhou population which was the highest resistance to imidacloprid and thiamethoxam. These findings suggested the P450 mono-oxygenase and symbiotic fungi together affected ACP resistance to imidacloprid and thiamethoxam. In the future, we may use environmental G. pallescens and A. sclerotigenum to control or delay ACP resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Yi, Lei, He, Yi, Li, Dai and Hong.)

Cereal cyst nematodes are sedentary biotrophic endoparasites that secrete effector proteins into plant tissues to transit normal cells into specialized feeding sites and suppress plant defenses. To understand the function of nematode effectors in Heterodera avenae , here, we identified a calreticulin protein HaCRT1, which could suppress the cell death induced by Bax when expressed in Nicotiana benthamiana . HaCRT1 is synthetized in the subventral gland cells of pre-parasitic second-stage nematodes. Real-time PCR assays indicated that the expression of HaCRT1 was highest in parasitic second-stage juveniles. The expression of an HaCRT1-RFP fusion in N. benthamiana revealed that it was localized in the endoplasmic reticulum of the plant cell. The ability of H. avenae infecting plants was significantly reduced when HaCRT1 was knocked down by RNA interference in vitro . Arabidopsis thaliana plants expressing HaCRT1 were more susceptible than wild-type plants to Pseudomonas syringae . The induction of defense-related genes, PAD4 , WRKY33 , FRK1 , and WRKY29 , after treatment with flg22 was suppressed in HaCRT1 -transgenic plants. Also, the ROS accumulation induced by flg22 was reduced in the HaCRT1 -transgenic plants compared to wild-type plants. HaCRT1 overexpression increased the cytosolic Ca 2+ concentration in A. thaliana . These data suggested that HaCRT1 may contribute to the pathogenicity of H. avenae by suppressing host basal defense., (Copyright © 2020 Liu, Peng, Su, Liu, Huang, Dai and Peng.)

Brachypodium distachyon , as an effective model of cereal grains, is susceptible to most destructive cereal pathogens. Senescence associated gene 101 ( SAG101 ) has been studied extensively in Arabidopsis . SAG101 is one of the important regulators of plant immunity. However, no homologous genes of AtSAG101 were found in B. distachyon . In this study, the AtSAG101 gene was transformed into B. distachyon. Three transgenic plant lines containing the AtSAG101 gene were confirmed by PCR and GUS gene activity. There were fewer Puccinia brachypodii urediospores in the AtSAG101 -overexpressing plants compared to wild type plants. P. brachypodii biomass was obviously decreased in AtSAG101 transgenic plants. The length of infection hyphae and infection unit areas of P. brachypodii were significantly limited in transgenic plants. Moreover, there were small lesions in AtSAG101 transgenic plants challenged by Magnaporthe oryzae . Salicylic acid accumulation was significantly increased, which led to elevated pathogenesis-related gene expression in transgenic B. distachyon inoculated by P. brachypodii or M. oryzae compared to wild type plants. These results were consistent with infected phenotypes. Overexpression of AtSAG101 in B. distachyon caused resistance to M. oryzae and P. brachypodii . These results suggest that AtSAG101 could regulate plant resistance in B. distachyon .

Begomoviruses can modify their transmission vector, Bemisia tabaci, to benefit their spread, although this may not always be the case. Here, the new begomovirus Ramie mosaic virus (RaMoV) and its vector B. tabaci MED, which is dominant in China and many regions of the world, were used as a model to examine direct and indirect interaction and virus transmission by B. tabaci MED of different sexes. No significant direct or indirect effects of RaMoV were observed in B. tabaci MED females, although RaMoV could shorten the life span of B. tabaci MED females by up to 4 days. A test of RaMoV transmission by different sexes of B. tabaci MED showed that there was higher virus transmission efficiency by females than males. Overall, RaMoV is transmitted by B. tabaci MED in a sex-dependent manner, and further research is needed to uncover the mechanism of the difference in RaMoV transmission by different sexes of B. tabaci.

The absorption of nutrients and disease resistance are two indispensable physiological processes in plants; however, it is still largely unknown whether there is cross-talk between their molecular signaling pathways. In this study, we identified the rice OsPT8 protein, which is a member of the phosphate transporters (PTs) Pht1 family and also plays a role in rice disease resistance. The transcriptional level of OsPT8 is suppressed after infection with rice pathogens and treatment with pathogen-associated molecular patterns (PAMPs). Overexpression of OsPT8 suppresses rice disease resistance against the pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. Accordingly, the transcription level of resistance related genes, such as PAL and PBZ1, is inhibited in plants overexpressing OsPT8 (OsPT8-OX) after inoculation with these pathogens. In OsPT8-OX plants, PAMPs-triggered immunity (PTI) response genes, such as OsRac1 and SGT1, are suppressed during treatment with PAMPs chitin or flg22. Moreover, the typical response of PTI is suppressed after chitin or flg22 treatment. We also identified OsPT8 as an interactor of a rice mitogen-activated protein kinase BWMK1, which is a regulator of disease resistance. Under low phosphate (Pi) conditions, the OsPT8-OX plants display better agronomic traits than the control plants. However, the differences in development between OsPT8-OX and the control plants are reduced upon the increase of Pi concentration. These results demonstrate that OsPT8 regulates the transduction of Pi signaling for development and negatively regulates rice immunity.

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated "Candidatus Liberibacter spp". "Candidatus Liberibacter spp" that infect Citrus includes "Candidatus Liberibacter asiaticus" (Las), "Candidatus Liberibacter africanus" (Laf) and "Candidatus Liberibacter americanus" (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection., Competing Interests: The authors have declared that no competing interests exist.

The various post-translational modifications (PTMs) of plant proteins have important regulatory roles in development. We therefore examined various modified proteins from strawberry stigmata and found that succinylation of lysine residues was the most abundant type of modification. We then subjected proteins from strawberry stigmata to an efficient enrichment method for succinylated peptides and identified 200 uniquely succinylated lysines (Suks) in 116 proteins. A bioinformatics analysis revealed that these proteins are involved in important biological processes, including stress responses, vesicular transport, and energy metabolism. Proteomics, combined with immunoprecipitation and immunoblotting, revealed an obvious increase in succinylation of the assembly polypeptide 2 (AP2) and clathrin from 0.5 to 2 h after pollination, suggesting that succinylation is involved in the recognition of pollen-stigma signaling substances and vesicular transport. These results suggest that AP2/clathrin-mediated vesicular transport processes are regulated by lysine succinylation during pollen recognition.

Huanglongbing (HLB), a systemic and destructive disease of citrus, is associated with ' Candidatus Liberibacter asiaticus' (Las) in the United States. Our earlier work has shown that Las bacteria were significantly reduced or eliminated when potted HLB-affected citrus were continuously exposed to high temperatures of 40 to 42 °C for a minimum of 48 h. To determine the feasibility and effectiveness of solar thermotherapy in the field, portable plastic enclosures were placed over commercial and residential citrus, exposing trees to high temperatures through solarization. Within 3-6 weeks after treatment, most trees responded with vigorous new growth. Las titer in new growth was greatly reduced for 18-36 months after treatment. Unlike with potted trees, exposure to high heat did not eradicate the Las population under field conditions. This may be attributed to reduced temperatures at night in the field compared to continuous high temperature exposure that can be maintained in growth chambers, and the failure to achieve therapeutic temperatures in the root zone. Despite the presence of Las in heat-treated commercial citrus, many trees produced abundant flush and grew vigorously for 2 to 3 years after treatment. Transcriptome analysis comparing healthy trees to HLB-affected citrus both before and after heat treatment demonstrated that post-treatment transcriptional expression patterns more closely resembled the expression patterns of healthy controls for most differentially expressed genes and that genes involved with plant-bacterium interactions are upregulated after heat treatment. Overall, these results indicate that solar thermotherapy can be an effective component of an integrated control strategy for citrus HLB., Competing Interests: The authors declare no conflict of interest.

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata , a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper , the Δ C t method, and RefFinder , were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata , our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Garlic virus X (GarVX) ORF3 encodes a p11 protein, which contributes to virus cell-to-cell movement and forms granules on the endoplasmic reticulum (ER) in Nicotiana benthamiana. Expression of p11 either from a binary vector, PVX or TMV induced ER stress and the unfolded protein response (UPR), as demonstrated by an increase in transcription of the ER luminal binding protein (BiP) and bZIP60 genes. UPR-related programmed cell death (PCD) was elicited by PVX : p11 or TMV : p11 in systemic infected leaves. Examination of p11 mutants with deletions of two transmembrane domains (TM) revealed that both were required for generating granules and for inducing necrosis. TRV-based VIGS was used to investigate the correlation between bZIP60 expression and p11-induced UPR-related PCD. Less necrosis was observed on local and systemic leaves of bZIP60 knockdown plants when infected with PVXp11, suggesting that bZIP60 plays an important role in the UPR-related PCD response to p11 in N. benthamiana.

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.

During their growth and development, plants are vulnerable to the effects of a variety of pathogens. Proteomics technology plays an important role in research studies of plant defense mechanisms by mining the expression changes of proteins in response to various biotic stresses. This review article provides a comprehensive overview of the latest developments in international proteomic research on plant biotic stress. It summarizes the methods commonly used in plant proteomic research to investigate biotic stress, analyze the protein responses of plants in adverse conditions, and reviews the applications of proteomics combined with transgenic technology in plant protection., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.

The nematophagous fungus Pochonia chlamydosporia, which belongs to the family Clavicipitaceae (Ascomycota: Pezizomycotina: Sordariomycetes: Hypocreales), is a promising biological control agent for root-knot and cyst nematodes. Its biocontrol effect has been confirmed by pot and field trials. The genome sequence of the fungus was completed recently; therefore, genome-wide functional analyses will identify its infection-associated genes. Gene knockout techniques are useful molecular tools to study gene functions. However, cultures of P. chlamydosporia are resistant to high levels of a range of fungal inhibitors, which makes the gene knockout technique difficult in this fungus. Fortunately, we found that the wild P. chlamydosporia strain PC-170 could not grow on medium containing 150μgml(-1) G418 sulfate, representing a new selectable marker for P. chlamydosporia. The neomycin-resistance gene (neo), which was amplified from the plasmid pKOV21, conferred G418-resistance on the fungus; therefore, it was chosen as the marker gene. We subsequently developed a gene knockout system for P. chlamydosporia using split-marker homologous recombination cassettes with resistance selection and protoplast transformation. The split-marker cassettes were developed using fusion PCR, and involved only two rounds of PCR. The final products comprised two linear constructs. Each construct contained a flanking region of the target gene and two thirds of the neo gene. Alkaline serine protease and chitinase were confirmed to be produced by P. chlamydosporia during infection of nematode eggs and could participate in lysis of the eggshell of nematode eggs. Here, we knocked out one chitinase gene, VFPPC&#95;01099, and two protease genes (VFPPC&#95;10088, VFPPC&#95;06535). We obtained approximately 100 suspected mutants after each transformation. After screening by PCR, the average rate of gene knockout was 13%: 11% (VFPPC&#95;01099), 13% (VFPPC&#95;10088) and 15% (VFPPC&#95;06535). This efficient and convenient technique will accelerate functional genomic studies in P. chlamydosporia., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Utilization of broad-spectrum resistance (R) genes is an effective and economical strategy to control the fungal pathogen Magnaporthe oryzae, the causal agent of the rice blast disease. Among the cloned blast resistance genes, Pi9, Pi2 and Piz-t confer broad-spectrum resistance to diverse M. oryzae isolates and were isolated from the Pi2/9 locus on chromosome 6. Identification and isolation of additional R genes with different resistance spectra from this locus will provide novel genetic resources for better control of this important rice disease., Results: In this study, we identified a dominant R gene, Pi2-2, at the Pi2/9 locus from Jefferson, an elite U.S. rice cultivar, through genetic and physical mapping. Inoculation tests showed that Jefferson has different resistant specificities to M. oryzae isolates compared rice lines with the Pi9, Pi2 and Piz-t genes. Fine mapping delimited Pi2-2 to a 270-kb interval between the markers AP5659-3 and RM19817, and this interval contains three nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the Nipponbare genome. Five bacterial artificial chromosome (BAC) clones spanning the region were identified, and a BAC contig covering the Pi2-2 locus was constructed., Conclusions: We identified a new allelic gene at the Pi2/9 locus and fine-mapped the gene within a 270-kb region. Our results provide essential information for the isolation of the Pi2-2 gene and tightly linked DNA markers for rice blast resistance breeding.

The ubiquitination pathway is involved in a variety of cellular processes in plant growth, development, and immune responses. However, the function of this pathway in connecting plant development and innate immunity is still largely unknown. Recently, we characterized the U-box/ARM E3 ubiquitin ligase PUB13, which regulates both immune responses and flowering time in Arabidopsis. Here, we show that the rice Spl11 gene can complement the cell death and flowering functions of PUB13 in the pub13 mutant. In addition, HFR1, which functions mainly in photomorphogenesis, was identified as one of the PUB13-interacting proteins through yeast two-hybrid screening and pull-down assays. Because the flowering phenotype of pub13 depends on photoperiod, we propose that PUB13 may regulate HFR1 to fine-tune photomorphogenesis and flowering time in Arabidopsis.

Tianjingyeshengdao' (TY) is a rice cultivar with durable resistance to populations of Magnaporthe oryzae (the causal agent of blast) in China. To understand the genetic basis of its resistance to blast, we developed a population of recombinant inbred lines from a cross between TY and the highly susceptible 'CO39' for gene mapping analysis. In total, 22 quantitative trait loci (QTLs) controlling rice blast resistance were identified on chromosomes 1, 3, 4, 5, 6, 9, 11, and 12 from the evaluation of four disease parameters in both greenhouse and blast nursery conditions. Among these QTLs, 19 were contributed by TY and three by CO39. Two QTL clusters on chromosome 6 and 12 were named Pi2-1 and Pi51(t), respectively. Pi2-1 was detected under both growth chamber and natural blast nursery conditions, and explained 31.24 to 59.73% of the phenotypic variation. Pi51(t) was only detected in the natural blast nursery and explained 3.67 to 10.37% of the phenotypic variation. Our results demonstrate that the durable resistance in TY is controlled by two major and seven minor genes. Identification of the markers linked to both Pi2-1 and Pi51(t) in this study should be useful for marker-aided selection in rice breeding programs as well as for molecular cloning of the identified resistance genes.

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDIS1 (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O. sativa). The expression of OsDIS1 was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDIS1 possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDIS1 was localized predominantly in the nucleus. Overexpression of OsDIS1 reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDIS1 enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDIS1 overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDIS1 interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDIS1 via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDIS1(H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDIS1 plays a negative role in drought stress tolerance through transcriptional regulation of diverse stress-related genes and possibly through posttranslational regulation of OsNek6 in rice.

The indica rice cultivar Xiangzi 3150 (XZ3150) confers a high level of resistance to 95% of the isolates of Magnaporthe oryzae (the agent of rice blast disease) collected in Hunan Province, China. To identify the resistance (R) gene(s) controlling the high level of resistance in this cultivar, we developed 286 F(9) recombinant inbred lines (RILs) from a cross between XZ3150 and the highly susceptible cultivar CO39. Inoculation of the RILs and an F(2) population from a cross between the two cultivars with the avirulent isolate 193-1-1 in the growth chamber indicated the presence of two dominant R genes in XZ3150. A linkage map with 134 polymorphic simple sequence repeat and single feature polymorphism markers was constructed with the genotype data of the 286 RILs. Composite interval mapping (CIM) using the results of 193-1-1 inoculation showed that two major R genes, designated Pi47 and Pi48, were located between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. Interestingly, the CIM analysis of the four resistant components of the RILs to the field blast population revealed that Pi47 and Pi48 were also the major genetic factors responsible for the field resistance in XZ3150. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars.

Ethylene response factor (ERF) proteins play important roles in regulating plant stress response and development. Our previous studies have shown that JERF3 activates the expression of oxidative stress responsive genes in transgenic tobacco and enhances tolerance to salt, drought, and freezing, indicating that JERF3 is a very important transcriptional regulator in dicot plants. In the study reported here, we further addressed the regulatory function of JERF3 in a monocot, rice, by generating transgenic rice plants overexpressing JERF3 and comparing these with non-transgenic rice plants for physiological and molecular alterations and tolerance to drought and osmotic stresses. Growth and development under normal growth conditions were the same in both the transgenic and non-transgenic rice. Interestingly, the JERF3 transgenic plants exhibited better stress tolerance, whereas the non-transgenic rice seedlings showed serious stress symptoms and ultimately died after the drought and osmotic treatments. Biochemical analysis revealed that the contents of soluble sugars and proline were significantly increased in transgenic rice compared with non-transgenic plants under dehydration conditions. In addition, overexpression of JERF3 in rice led to the up-regulated expression of two OsP5CS genes in response to drought treatment compared with their expression in non-transgenic plants. JERF3 also activated the expression of stress-responsive genes, including WCOR413-like, OsEnol, and OsSPDS2, in transgenic rice under normal growth conditions. These data suggest that JERF3 plays important roles in transgenic rice and that it is likely to be beneficial in engineering crop plants with improved tolerance to drought and osmotic stresses.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is the most devastating disease of rice and severely affects crop stability and sustainability worldwide. This disease has advanced to become one of the premier model fungal pathosystems for host-pathogen interactions because of the depth of comprehensive studies in both species using modern genetic, genomic, proteomic and bioinformatic approaches. Many fungal genes involved in pathogenicity and rice genes involved in effector recognition and defence responses have been identified over the past decade. Specifically, the cloning of a total of nine avirulence (Avr) genes in M. oryzae, 13 rice resistance (R) genes and two rice blast quantitative trait loci (QTLs) has provided new insights into the molecular basis of fungal and plant interactions. In this article, we consider the new findings on the structure and function of the recently cloned R and Avr genes, and provide perspectives for future research directions towards a better understanding of the molecular underpinnings of the rice-M. oryzae interaction.

Plants employ multifaceted mechanisms to fight with numerous pathogens in nature. Resistance (R) genes are the most effective weapons against pathogen invasion since they can specifically recognize the corresponding pathogen effectors or associated protein(s) to activate plant immune responses at the site of infection. Up to date, over 70 R genes have been isolated from various plant species. Most R proteins contain conserved motifs such as nucleotide-binding site (NBS), leucine-rich repeat (LRR), Toll-interleukin-1 receptor domain (TIR, homologous to cytoplasmic domains of the Drosophila Toll protein and the mammalian interleukin-1 receptor), coiled-coil (CC) or leucine zipper (LZ) structure and protein kinase domain (PK). Recent results indicate that these domains play significant roles in R protein interactions with effector proteins from pathogens and in activating signal transduction pathways involved in innate immunity. This review highlights an overview of the recent progress in elucidating the structure, function and evolution of the isolated R genes in different plant-pathogen interaction systems.

The broad-spectrum rice blast resistance gene Pi9 was cloned using a map-based cloning strategy. Sequencing of a 76-kb bacterial artificial chromosome (BAC) contig spanning the Pi9 locus led to identification of six tandemly arranged resistance-like genes with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) (Nbs1-Pi9-Nbs6-Pi9). Analysis of selected Pi9 deletion mutants and transformation of a 45-kb fragment from the BAC contig into the susceptible rice cultivar TP309 narrowed down Pi9 to the candidate genes Nbs2-Pi9 and Nbs3-Pi9. Disease evaluation of the transgenic lines carrying the individual candidate genes confirmed that Nbs2-Pi9 is the Pi9 gene. Sequence comparison analysis revealed that the six paralogs at the Pi9 locus belong to four classes and gene duplication might be one of the major evolutionary forces contributing to the formation of the NBS-LRR gene cluster. Semiquantitative reverse transcriptase (RT)-PCR analysis showed that Pi9 was constitutively expressed in the Pi9-resistant plants and was not induced by blast infection. The cloned Pi9 gene provides a starting point to elucidate the molecular basis of the broad-spectrum disease resistance and the evolutionary mechanisms of blast resistance gene clusters in rice.

The F-box protein gene COI1 from Arabidopsis plays a fundamental role in response to jasmonates, which regulate plant root growth, pollen fertility, wounding and healing, and defense against pathogens and insects. Null mutations in COI1 were previously found to abolish all the jasmonate responses, and the Arabidopsis coil-1 mutant is male sterile and susceptible to pathogen infection. In this study, we isolated an F-box protein gene from soybean, which shares significant homology with the Arabidopsis COI1 and similarly contains an F-box motif and leucine rich repeats (LRR), here designated GmCOI1 (Glycine max L. (Merr.) COI1). To test whether the sequence homology and structural similarity are indicative of functional conservation, we expressed GmCOI1 in the Arabidopsis coil-1 mutant. The transgenic coil-1 plants with expression of the GmCOI1 gene were found to exhibit normal jasmonate responses, including jasmonate-regulated plant defense and fertility. In addition, the chimerical proteins with swapped domain of the F-box motif or LRR between GmCOI1 and COI1 were shown to functionally complement the coil-1 mutation. Furthermore, GmCOI1 was found to assemble into the Skpl-Cullin-F-box (SCF) complexes, similar to the formation of the Arabidopsis SCF(COO1). These data demonstrate the soybean F-box protein gene GmCOI1 is able to mediate jasmonate-regulated plant defense and fertility in Arabidopsis, which implies a generic jasmonate pathway with conserved signal components in different plant species.

The Arabidopsis thaliana CORONATINE INSENSITIVE1 (COI1) gene encodes an F-box protein to assemble SCF(COI1) complexes essential for response to jasmonates (JAs), which are a family of plant signaling molecules required for many essential functions, including plant defense and reproduction. To better understand the molecular basis of JA action, we screened for suppressors of coi1 and isolated a coi1 suppressor1 (cos1) mutant. The cos1 mutation restores the coi1-related phenotypes, including defects in JA sensitivity, senescence, and plant defense responses. The COS1 gene was cloned through a map-based approach and found to encode lumazine synthase, a key component in the riboflavin pathway that is essential for diverse yet critical cellular processes. We demonstrated a novel function for the riboflavin pathway that acts downstream of COI1 in the JA signaling pathway and is required for suppression of the COI1-mediated root growth, senescence, and plant defense.

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. The COI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with the Arabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action in planta. Functional analysis by antisense strategy showed that ASK1 is involved in male fertility.