Gene characterization of Bradyrhizobium spp. strains contrasting in biological nitrogen fixation efficiency in soybean

Bacteria from genus Bradyrhizobium can establish symbiosis with soybean and supply the plant nitrogen demands via biological nitrogen fixation (BNF). This study aimed to characterize genes related to BNF efficiency in B. japonicum strains contrasting in BNF efficiency. These gene sequences were previously identified in B. japonicum (strain S370) as probably related to the BNF efficiency in soybean using a DNA subtractive technique. These genes were amplified with primers based on B. japonicum USDA110 genome. The PCR products were digested with restriction endonucleases and the RFLP products were analyzed by horizontal electrophoresis. Among the four genes, only blr3208 and blr4511 amplified for most of the strains. Neither polymorphism of the restriction profile of blr3208 and blr4511 genes nor with endonuclease for PCR-RFLP was observed. The contrasting strains had blr3208 and blr4511 genes sequenced and the multiple alignment analysis of nucleotide sequences showed the presence of preserved internal regions, confirming the analysis with PCR-RFLP. The blr3208 and blr4511 genes are highly conserved among B. japonicum strains, which may be related to adaptive function during the evolutionary process of Bradyrhizobium genus.

A putative effector UvHrip1 inhibits BAX-triggered cell death in Nicotiana benthamiana, and infection of Ustilaginoidea virens suppresses defense-related genes expression

Rice false smut (RFS), caused by Ustilaginoidea virens, is one of the most detrimental rice fungal diseases and pose a severe threat to rice production and quality. Effectors in U. virens often act as a set of essential virulence factors that play crucial roles in the interaction between host and the pathogen. Thus, the functions of each effector in U. virens need to be further explored. Here, we performed multiple alignment analysis and demonstrated a small secreted hypersensitive response-inducing protein (hrip), named UvHrip1, was highly conserved in fungi. The predicted SP of UvHrip1 was functional, which guided SUC secreted from yeast and was recognized by plant cells. The localization of UvHrip1 was mainly in the nucleus and cytoplasm monitored through the GFP fusion protein in Nicotiana benthamiana cells. uvhrip1 was drastically up-regulated in the susceptible cultivar LYP9 of rice during the pathogen infection, while did not in the resistant cultivar IR28. We also proved that UvHrip1 suppressed the mammalian BAX-induced necrosis-like defense symptoms in N. benthamiana. Furthermore, patterns of expression of defense-related genes, OsPR1#012 and OsPR10b, were regulated over U. virens infection in rice. Collectively, our data demonstrated that infection of U. virens suppresses defense-related genes expression and UvHrip1 was most likely a core effector in regulating plant immunity.

Structural Similarity Analysis of Spike Proteins of SARS-CoV-2 and Other SARS-related Coronaviruses

Objectives Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high infectivity in humans, attributed to the strong affinity of its spike (S) protein to human angiotensin-converting enzyme 2 (ACE2). Here, we analyzed the structural similarity of the S protein between SARS-CoV-2 and other SARS-related coronaviruses (CoVs). Methods We performed multiple alignment analysis of nine amino acid sequences of CoV S proteins from NCBI with MAFFT web-based software, followed by phylogeny analysis. Three-dimensional structure modeling was performed by SWISS-MODEL. We calculated the template modeling score between the S protein of SARS-CoV-2 and that of other SARS-related CoVs. Results The S1 domain of the unclassified CoV RaTG13 (the host of which is the intermediate horseshoe bat) was structurally very similar to that of SARS-CoV-2, implying that RaTG13 could be the origin of SARS-CoV-2. In addition, the folding property of the entire S protein was nearly the same between SARS-CoV-2 and RaTG13 after the PRRA amino acid insertion was removed from SARS-CoV-2. Conclusions RaTG13 could have a high binding affinity to ACE2, similar to SARS-CoV-2, and it is therefore highly likely to infect other animals. Therefore, massive research and monitoring of CoVs in animals is necessary to prevent future COVID-19-like disasters.

MYB transcription factor CiMYB42 regulates limonoids biosynthesis in citrus

Backgrounds Limonoids, a major bioactive components, are produced by triterpenoids metabolic pathway. So far, the detailed biochemical reactions regarding to limonoid biosynthesis and their molecular regulation remain elusive. The identification of transcription factors that regulate limonoids biosynthetic pathways is not only necessary for understanding the regulatory mechanisms but also as a tool for manipulating biosynthetic genes for biotechnological applications.Results In this study, CiMYB42 transcription factor was isolated and identified. Multiple alignment analysis and phylogenetic analysis demonstrated that CiMYB42 is a typical R2R3MYB transcription factor and its amino acid is similar to that of AtMYB42 . The limonoids content was higher in the citrus sinensis and citrus grandis than other species. The diverse accumulation patterns also showed in different leaf developmental stages. Expression of CiMYB42 was significantly related to limonoids content and the expression of CiOSC in some of citrus varieties. CiMYB42 transgenic sweet orange resulted significantly change on limonoids contents. Noticeably, CiMYB42 -RNAi induced dwarf phenotype and mainly decreased nomilin accumulation. Overexpressing CiMYB42 mainly increased limonin content. Yeast one hybrid assay results indicated that CiMYB42 exclusively bind to the promoter of CiOSC. In brief, CiMYB42 involved in the limonoids biosynthesis through binding the promoter of CiOSC in citrus.Conclusions These results indicated that CiMYB42 is an important transcription activator involved in limonoids biosynthesis by regulating the expression of CiOSC . This is the first report elucidating the role of transcription factor in citrus limonoids biosynthesis. Our contributions will provide a reference to understanding regulatory mechanisms of R2R3MYB TFs in the triterpenoids biosynthetic pathway.

Molecular characterisation and expression analysis of scavenger receptor class B member 1 (SR-B1) gene in Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

The scavenger receptor class B member 1 (SR-B1) plays an important role in the first level of host defense against invading pathogens. In the present study, we cloned and characterized SR-B1 gene from Oreochromis niloticus (Linnaeus, 1758). The sequence of SR-B1 is 2248-bp long and contains a 1404-bp ORF encoded 467 amino acids. The sequence alignment showed that SR-B1 gene contains ten exons and nine introns. A multiple alignment analysis suggested that SR-B1 protein contains the conserved CXXS redox and GXXXG motifs, and shared high similarities with that of other species. On a phylogenetic tree, SR-B1 clustered with those of other teleosts and formed a separate fish clade. Real-time quantitative PCR analysis showed that SR-B1 was differentially expressed in various tissues and the expression levels in spleen and intestine were significantly upregulated by Streptococcus agalactiae infection. The results suggest that SR-B1 may be involved in the immune response against bacteria in Nile tilapia.

Detection and Characterization of QoI-Resistant Phytophthora capsici Causing Pepper Phytophthora Blight in China

Phytophthora capsici is a highly destructive plant pathogen that has spread worldwide. To date, the quinone outside inhibitor (QoI) azoxystrobin has been the choice of farmers for managing this oomycete. In this study, the sensitivity of 90 P. capsici isolates collected from Yunnan, Fujian, Jiangxi, Zhejiang, and Guangdong in southern China to azoxystrobin was assessed based on mycelial growth, sporangia formation, and zoospore discharge. Furthermore, the mitochondrial cytochrome b (cytb) gene from azoxystrobin-sensitive and -resistant P. capsici isolates was compared to investigate the mechanism of QoI resistance. The high values for effective concentration to inhibit 50% of mycelial growth and large variation factor obtained provide strong support for the existence of azoxystrobin-resistant subpopulations in wild populations. The resistance frequency of P. capsici to azoxystrobin was greater than 40%. Sensitive P. capsici isolates were strongly suppressed on V8 medium plates containing azoxystrobin supplemented with salicylhydroxamic acid at 50 µg ml−1, whereas resistant isolates grew well under these conditions. Multiple alignment analysis revealed a missense mutation in the cytb gene that alters codon 137 (GGA to AGA), causing an amino acid substitution of glycine to arginine (G137R). The fitness of the azoxystrobin-sensitive isolate is similar to that of the G137R mutant. Additionally, the P. capsici isolates used in this study exhibited decreased sensitivity to two other QoI fungicides (pyraclostrobin and famoxadone). Necessary measures should be taken to control this trend of resistance to QoI that has developed in P. capsici in southern China.

cytbas a New Genetic Marker for Differentiation ofProtothecaSpecies

ABSTRACTAchlorophyllous unicellular microalgae of the genusPrototheca(Trebouxiophyceae,Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused byProtothecawickerhamii, followed byProtothecazopfiiand, sporadically, byProtothecacutisandProtothecamiyajii. In veterinary medicine, however,P. zopfiiis a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification ofProtothecaspp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among allProtothecaspp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of allProtothecaspp. We propose the mitochondrialcytbgene as a new and robust marker for diagnostics and phylogenetic studies of theProtothecaalgae. Thecytbgene displayed important advantages over the rDNA markers. Not only did thecytbgene have the highest discriminatory capacity for resolving allProtothecaspecies, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partialcytbgene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of allProtothecaspecies described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Cloning and Expression Analysis of a Farnesyl Diphosphate Synthase (FPPS) Gene from Chamaemelum nobile

Farnesyl diphosphate synthase (FPPS), an isopentenyl transferase, catalyzes the condensation reaction of five carbon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form fifteen carbon farnesyl pyrophosphate (FPP), which is the key precursor for sesquiterpene biosynthesis. In this study, a FPPS gene (CnFPPS) was cloned from Chamaemelum nobile. The full-length cDNA of CnFPPS is 1239 bp and contains an open reading frame (ORF) of 1029 bp encoding 342 amino acids. The theoretical molecular weight and pI of the CnFPPS protein are 39.38 kDa and 5.59, respectively. Multiple alignment analysis showed the protein sequence of CnFPPS had a high homology with FPPS proteins from other plants. The deduced amino acid of CnFPPS contained five conservative domains such as substrate binding pocket, substrate-Mg2+ binding site, catalytic site, aspartate-rich region 1 and 2, suggesting CnFPPS is one member of FPPS family in C. nobile. Phylogenetic analysis based on the amino acid sequences of FPPSs showed that CnFPPS was closely related to the FPPS of Matricaria chamomilla. The result of qRT-PCR revealed that CnFPPS gene was constitutively expressed in different tissues of C. nobile, with the highest expression in the root. These findings improve the understanding of the synthesis and regulation of the terpenoid compounds at the molecular level and lay a foundation for studying the regulatory functions of CnFPPS in terpenoid biosynthetic pathway in C. nobile.

Cloning and expression of delta-1-pyrroline-5-carboxylate dehydrogenase in Escherichia coli DH5α improves phosphate solubilization

A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5′ end of the RNA transcript (SMART) technique. A total of 106 clones showed halos in tricalcium phosphate (TCP) medium, and clone I-40 showed clear halos. The full-length cDNA of clone I-40 was 1355 bp with a complete open reading frame (ORF) of 1032 bp, encoding a protein of 343 amino acids. Multiple alignment analysis revealed a high degree of homology between the ORF of clone I-40 and delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) of other fungi. The ORF expression vector was constructed and transformed into Escherichia coli DH5α. The transformant (ORF-1) with the P5CDH gene secreted organic acid in medium with TCP as the sole source of phosphate. Acetic acid and α-ketoglutarate were secreted in 4 and 24 h, respectively. ORF-1 decreased the pH of the medium from 6.62 to 3.45 and released soluble phosphate at 0.172 mg·mL−1 in 28 h. Expression of the P. oxalicum I1 p5cdh gene in E. coli could enhance organic acid secretion and phosphate-solubilizing ability.