Identification of Resistance to Pathogenesis Related Protein GmPR1L in Tobacco Botrytis cinerea Infection

Soybean(Glycine max(Linn.)Merr.)annual leguminous crop is cultivated all over the world.The occurrence of diseases has a great impact on the yield and quality of soybean.In this study,based on the RNA-seq of soybean variety M18,a complete CDS(Coding sequence)GmPR1L of the pathogenesis-related protein 1 family was obtained,which has the ability to resist fungal diseases.The overexpression vector and interference expression vector were transferred into tobacco NC89,and the resistance of transgenic tobacco(Nicotiana tabacum L.)to Botrytis cinerea infection was identified.The results show that:Compared with the control,the activities of related defense enzymes SOD(Superoxide dismutase),POD(Peroxidase),PAL(L-phenylalanine ammonia-lyase)and PPO(Polyphenol oxidase)in the over-expressed transgenic tobacco OEA1 and OEA2 increased to different degrees,and increased significantly at different infection time points.The activities of defense enzymes in the interfering strains IEA1 and IEA2 were significantly lower than those in the control strains.The results of resistance level identification showed that the disease spot rate of OEA1 was significantly lower than that of the control line,and the disease spot rate of OEA2 was significantly lower than that of the control line.The plaque rate of the interfering expression line IEA1-IEA2 was significantly higher than that of the control line.It is preliminarily believed that the process related protein GmPR1L can improve the resistance of tobacco to B.cinerea.

GmSKP1,a Novel S-phase Kinase-associated Protein 1 in Glycine max,Enhancing Resistance Against Phytophthora sojae Infection

Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins are key members of the SKP1/Cullin/F-box protein(SCF)ubiquitin ligase complex and play diverse roles in plant biology.However,the role of SKP1 in soybean against the phytopathogenic oomycete P.sojae remains unclear.In this study,a novel member of the soybean SKP1 gene family,GmSKP1 which was significantly induced by P.sojae,was reported.The expression of GmSKP1 was simultaneously induced by methyl jasmonate(MeJA),salicylic acid(SA)and ethylene(ET),which might suggest an important role for GmSKP1 of plant in responses to hormone treatments.Functional analysis using GmSKP1 overexpression lines showed that GmSKP1 enhanced resistance to P.sojae in transgenic soybean plants.Further analyses showed that GmSKP1 interacted with a homeodomain-leucine zipper protein transcription factor(GmHDL56)and a WRKY transcription factor(GmWRKY31),which could positively regulate responses to P.sojae in soybean.Importantly,several pathogenesis-related(PR)genes were constitutively activated,including GmPR1a,GmPR2,GmPR3,GmPR4,GmPR5a and GmPR10,in GmSKP1-OE soybean plants.Taken together,these results suggested that GmSKP1 enhanced resistance to P.sojae in soybean,possibly by activating the defense-related PR genes.

The G-protein α subunit GhGPA positively regulates Gossypium hirsutum resistance to Verticillium dahliae via induction of SA and JA signaling pathways and ROS accumulation

Verticillium wilt, a devastating disease in cotton caused by Verticillium dahliae, reduces cotton quality and yield. Heterotrimeric GTP-binding proteins, consisting of Ga, Gb, and Gc subunits, transducers of receptor signaling, function in a wide range of biological events. However, the function of Ga proteins in the regulation of defense responses in plants is largely unexplored, except for a few reports on model species. In the present study, a cotton G-protein a-subunit-encoding gene(GhGPA) was isolated from Verticillium wilt-resistant Gossypium hirsutum(upland cotton) cv. ND601. GhGPA transcription was up-regulated under V. dahliae stress, with higher expression in tolerant than in susceptible cotton cultivars.Subcellular localization revealed GhGPA to be located in the plasma membrane. GhGPA shows high(85.0%) identity with Arabidopsis AT2 G26300(AtGPA1), and AtGPA1 gpa1-4 mutants displayed susceptibility to V. dahliae. Ectopic expression of GhGPA successfully restored the resistance of Arabidopsis gpa1-4 mutants to Verticillium wilt and made them more resistant than the wild type. Overexpression of GhGPA in Arabidopsis markedly increased the resistance and resulted in dramatic up-regulation of pathogenesis-related(PR) genes and increased in H2 O2 accumulation and salicylic acid(SA) and jasmonic acid(JA) contents. However, suppressing GhGPA expression via virus-induced gene silencing(VIGS)increased susceptibility to Verticillium wilt, down-regulated the expression of PR and marker genes in SA and JA signaling pathways, and reduced H2 O2 content. The contents of SA and JA in Arabidopsis gpa1-4 and VIGS cotton were lower than those in the wild type and empty-vector control. However,GhGPA-overexpressing Arabidopsis contained more SA and JA than the wild type when inoculated with V. dahliae. Thus, GhGPA plays a vital role in Verticillium wilt resistance by inducing SA and JA signaling pathways and regulating the production of reactive oxygen species. These findings not only broaden our knowledge about the biological role of GhGPA, but also shed light on the defense mechanisms involving GhGPA against V. dahliae in cotton.

Analysis of Codon Usage Pattern of Banana Basic Secretory Protease Gene

[Objective] The objective of this study was to understand the codon usage bias pattern of banana pathogenesis-related 17 gene, Basic Secretory Protease gene(MaBSP). [Method] Relative codon usage patterns of MaBSP were calculated using the software CodonW version 1.4.2. and the web-based tool(http://kazusa.or.jp/codon/).[Result] Our findings showed that C-ended and G-ended codons were the most preferential except the TER codon UGA which was coded for by just one codon. The ENc value, relationship between AT bias and GC bias, Random synonymous codon usage(RSCU) and CAI all showed that codon bias usage existed in MaBSP gene.[Conclusion] The codon usage patterns of MaBSP gene is principally influenced by natural selection in the third position. However, other multiple factors also influence this pattern.

Role of Pathogen-Related Protein 10 (PR 10) under Abiotic and Biotic Stresses in Plants

Members of the Pathogenesis Related(PR)10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them.PR10 protein possesses ribonuclease(RNase)activity,interacts with phytohormones,involved in hormone-mediated signalling,afforded protection against various phytopathogenic fungi,bacteria,and viruses particularly in response to biotic and abiotic stresses.The resistance mechanism of PR10 protein may include activation of defense signalling pathways through possible interacting proteins involved in mediating responses to pathogens,degradation of RNA of the invading pathogens.Moreover,several morphological changes have been shown to accompany the enhanced abiotic stress tolerance.In this review,the possible mechanism of action of PR10 protein against biotic and abiotic stress has been discussed.Furthermore,our findings also confirmed that the in vivo Nitric oxide(NO)is essential for most of environmental abiotic stresses and disease resistance against pathogen infection.The proper level of NO may be necessary and beneficial,not only in plant response to the environmental abiotic stress,but also to biotic stress.The updated information on this interesting group of proteins will be useful in future research to develop multiple stress tolerance in plants.

Effects of Agro-antibiotic 211 on Related Substances Inducing Rice Resistance to Sheath Blight

[Objective] The paper was to explore the effect of agro-antibiotic 211, the active substance produced by streptomyces JD211, on rice resistance to sheath blight. [Method] Through pot experiment, the induction effect of agro-antibiotic 211 on rice resistance against sheath blight was analyzed, and the effect on phenolic metabolites and pathogenesis-related proteins were studied.[Result] The total phenol, phenylalanine ammonialyase, lignin, chitinase and β-1, 3-glucanase of rice leaves treated by agro-antibiotic 211 were slightly higher than that in control. At 96 h post treatment, the activity of β-1, 3-glucanase was 36.84% and 10.48% higher than that of blank control and CK+ Rhizoctonia solani, respectively; at120 h post treatment, the activity of phenylalanine ammonia-lyase were 42.13% and 62.28% higher than that of blank control and CK+ R. solani,respectively. The effect of agro-antibiotic 211 + R. solani was equal or slightly higher than that of Jinggangmycin + R. solani. [Conclusion] Agroantibiotic 211 induced rice resistance against sheath blight, and was closely related to the accumulation of phenolic substances and the increase of pathogenesis-related proteins.

Functional Analysis of a Wilt Fungus Inducible <i>PR</i>10-1 Gene from Cotton

Early stage expression of PR10 combined with phytoalexins contributed to Verticillium wilt resistance in cotton. In order to analysis the activities of PR10 proteins during pathogens’ infection, we cloned a Verticillium-induced PR10 (GbPR10-1) gene from cotton (Gossypium barbadense) and compared its expression patterns and domains with other PR10 proteins. Bioinformatics indicated that GbPR10-1 showed the lowest similarity with other 12 different PR10 genes in cotton (Upland and sea-island cotton). Expression profiles showed that GbPR10-1 gene instantly up-regulated after infection by V. dahliae in the sea-island cotton plants. GbPR10-1 was also induced by environmental stimulus including heat, submergence and salt, and ethylene but not by ABA and salicylic acid. The GbPR10-1 protein expressed in E. coli BL21 demonstrated that it had a low ribonuclease-like activity in vitro, and could inhibit V. dahliae hyphae growth but not its spores. Comparison analysis of GbPR10-1 (from resistant species) and GhPR10-1 (from susceptible species) responding to V. dahliae infection, only GbPR10-1 gene was strongly induced in the sea-island cotton plants (incompatible response), indicating that PR10-1 genes was linked to resistance signal. In summary, the earlier activation of GbPR10-1 gene, as the index of resistance response, would be aid to block

Comparative Proteomics Reveals Set of Oxidative Stress and Thaumatin-Like Proteins Associated with Resistance to Late Blight of Tomato

Proteomics techniques were used to study the molecular mechanisms involved in the defense of tomato against late blight (Phytophthora infestans). Proteins were extracted from resistant access BGH-2127 and susceptible cultivar “Santa Clara”. Leaves of the inoculated and non-inoculated (control) genotypes were collected at 0, 2, and 48 h after inoculation and analyzed by two-dimensional electrophoresis (2-DE), followed by identification with mass spectrometry (MALDI TOF-TOF). A total of 56 differentially abundant proteins were identified, of which 39 were resistant genotypes and 17 were susceptible. These proteins were categorized into functional groups of energy and metabolism, photosynthesis, stress and defense, transcription, other proteins, and as un-characterized ones. For access BGH-2127, oxidative stress proteins (2-cis peroxiedoxin BAS1 and 2-cis peroxiredoxin) and thaumatin-like protein showed increase in the relative abundance at 0 and 48 h of inoculation, respectively, and were therefore considered important for the defense mechanism of this genotype. The expression standards evaluated by real-time PCR differed from the results of the proteomic analysis. The protein-protein interaction networks provided important information on the cellular activities involved in the resistance of BGH-2127 late blight.

Tomato SlPti5 plays a regulative role in the plant immune response against Botrytis cinerea through modulation of ROS system and hormone pathways

While SlPti5 has been shown to play a crucial role in the regulation of antagonistic genes in Solanum lycopersicum and Arabidopsis against pathogen infection,there have been no comprehensive studies on the effects of SlPti5 on the regulatory response mechanism of reactive oxygen species(ROS) system and hormone pathways during growth and disease resistance of tomato plants.Here,we investigated the function of SlPti5 in the defense response of tomato against Botrytis cinerea utilizing a virus-induced gene silencing(VIGS)-based system.Expression profile analysis showed that SlPti5 was significantly induced upon B.cinerea infection,with high expression levels in the leaves and fruit of tomato.VIGS-based silencing of SlPti5 inhibited early vegetative growth,increased the plant’s susceptibility to infection,promoted the development of ROS,affected the expression of genes involved in the ROS scavenging system,and attenuated the expression of genes associated with pathogenesis and the ethylene/jasmonic acid signaling pathways.In sum,our data demonstrated that SlPti5 stimulates the immune response of tomato plant to Botrytis cinerea infection by involving the ethylene(ET)-and jasmonic acid(JA)-mediated pathways and modulating the expression of some key pathogenesis-related(PR) genes.

Presence of Virulence-Associated Genes and Ability to Form Biofilm among Clinical Isolates of <i>Escherichia coli</i>Causing Urinary Infection in Domestic Animals

Background: Urinary tract infection caused by Escherichia coli is a frequently observed condition both in humans and animals. Uropathogenic E. coli (UPEC) has been shown to have a pathogenicity island that enables them to infect the urinary tract. Because there is little information about the presence of UPEC-associated virulent genes in animal isolates this work was carried out with the intent to enhance the understanding about the strains of E.coli that cause infections in animals. Results: We screened 21 E. coli strains isolated causing urinary tract infection in domestic animals. Primers were designed to amplify urinary infection-associated genes. Nine genes, papA, tcpC, fyuA, tpbA, Lma, hylA, picU, tonB, and flicC were then amplified and sequenced. Different from the human isolate CFT073, all the animals E. coli lack some of the pathogenesis-associated genes. Genes encoding for proteins used to scavenge iron appear not to be so necessary during animal infections as they are in human infection. In further investigation of phenotypic properties, it was observed that animal UPECs have significantly more impaired ability to form biofilms than human UPEC strain. Conclusions: This study identified significant differences between human and animal UPECs. This may have its roots in the fact that it is difficult to determine if an animal has symptoms. Future studies will focus on some of the observations.