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873100.9723Isolation of Potato Endophytes and Screening of Chaetomium globosum Antimicrobial Genes. Antimicrobial peptides (AMPs) have natural antibacterial activities that pathogens find difficult to overcome. As a result of this occurrence, AMPs can act as an important substitute against the microbial resistance. In this study, we used plate confrontation tests to screen out 20 potential endophytes from potato tubers. Among them, endophyte F5 was found to significantly inhibit the growth of five different pathogenic fungi. Following that, phylogenetic analysis revealed that the internal transcribed spacer (ITS) sequences were 99% identical to Chaetomium globosum corresponding sequences. Thereafter, the Bacillus subtilis expression system was used to create a C. globosum cDNA library in order to isolate the resistance genes. Using this approach, the resistance gene screening technology in the indicator bacteria built-in library was used to identify two antimicrobial peptides, CgR2150 and CgR3101, with broad-spectrum antibacterial activities. Furthermore, the results showed that CgR2150 and CgR3101 have excellent UV, thermal, and enzyme stabilities. Also, these two peptides can significantly inhibit the growth of various bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Clavibacter michiganensis, and Clavibacter fangii) and fungi (Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea). Scanning electron microscopy (SEM) observations revealed that CgR2150 and CgR3101 peptides act against bacteria by disrupting bacterial cell membranes. Moreover, hemolytic activity assay showed that neither of the two peptides exhibited significant hemolytic activity. To conclude, the antimicrobial peptides CgR2150 and CgR3101 are promising in the development of a new antibacterial agent and for application in plant production.202235563004
1610.9708A glycoside hydrolase 30 protein BpXynC of Bacillus paralicheniformis NMSW12 recognized as A MAMP triggers plant immunity response. Bacillus spp. has been widely used as a biocontrol agent to control plant diseases. However, little is known about mechanisms of the protein MAMP secreted by Bacillus spp. Herein, our study reported a glycoside hydrolase family 30 (GH30) protein, BpXynC, produced by the biocontrol bacteria Bacillus paralicheniformis NMSW12, that can induce cell death in several plant species. The results revealed that the recombinant protein triggers cell death in Nicotiana benthamiana in a BAK1-dependent manner and elicits an early defense response, including ROS burst, activation of MAPK cascades, and upregulation of plant immunity marker genes. BpXynC was also found to be a glucuronoxylanase that exhibits hydrolysis activity on xlyan. Two mutants of BpXynC which lost the glucuronoxylanase activity still retained the elicitor activity. The qRT-PCR results of defense-related genes showed that BpXynC induces plant immunity responses via an SA-mediated pathway. BpXynC and its mutants could induce resistance in N. benthamiana against infection by Sclerotinia sclerotiorum and tobacco mosaic virus (TMV). Furthermore, BpXynC-treated tomato fruits exhibited strong resistance to the infection of Phytophthora capsica. Overall, our study revealed that GH30 protein BpXynC can induce plant immunity response as MAMP, which can be further applied as a biopesticide to control plant diseases.202438286384
1820.9694Antivirulence effects of cell-free culture supernatant of endophytic bacteria against grapevine crown gall agent, Agrobacterium tumefaciens, and induction of defense responses in plantlets via intact bacterial cells. BACKGROUND: Crown gall disease caused by Agrobacterium tumefaciens is a very destructive affliction that affects grapevines. Endophytic bacteria have been discovered to control plant diseases via the use of several mechanisms. This research examined the potential for controlling crown gall by three endophytic bacteria that were previously isolated from healthy cultivated and wild grapevines including Pseudomonas kilonensis Ba35, Pseudomonas chlororaphis Ba47, and Serratia liquefaciens Ou55. RESULT: At various degrees, three endophytic bacteria suppressed the populations of A. tumefaciens Gh1 and greatly decreased the symptoms of crown gall. Furthermore, biofilm production and motility behaviors of A. tumefaciens Gh1were greatly inhibited by the Cell-free Culture Supernatant (CFCS) of endophytic bacteria. According to our findings, CFCS may reduce the adhesion of A. tumefaciens Gh1 cells to grapevine cv. Rashe root tissues as well as their chemotaxis motility toward the extract of the roots. When compared to the untreated control, statistical analysis showed that CFCS significantly reduced the swimming, twitching, and swarming motility of A. tumefaciens Gh1. The findings demonstrated that the endophytic bacteria effectively stimulated the production of plant defensive enzymes including superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and total soluble phenols at different time intervals in grapevine inoculated with A. tumefaciens Gh1. The Ba47 strain markedly increased the expression levels of defense genes associated with plant resistance. The up-regulation of PR1, PR2, VvACO1, and GAD1 genes in grapevine leaves indicates the activation of SA and JA pathways, which play a role in enhancing resistance to pathogen invasion. The results showed that treating grapevine with Ba47 increased antioxidant defense activities and defense-related gene expression, which reduced oxidative damage caused by A. tumefaciens and decreased the incidence of crown gall disease. CONCLUSION: This is the first study on how A. tumefaciens, the grapevine crown gall agent, is affected by CFCS generated by endophytic bacteria in terms of growth and virulence features. To create safer plant disease management techniques, knowledge of the biocontrol processes mediated by CFCS during microbial interactions is crucial.202438336608
883130.9694Search for biocontrol agents among endophytic lipopeptide-synthesizing bacteria Bacillus spp. to protect wheat plants against Greenbug aphid (Schizaphis graminum). Beneficial endophytic bacteria can suppress the development of insect pests through direct antagonism, with the help of metabolites, or indirectly by the induction of systemic resistance through the regulation of hormonal signaling pathways. Lipopeptides are bacterial metabolites that exhibit direct antagonistic activity against many organisms, including insects. Also, lipopeptides are able to trigger induced systemic resistance (ISR) in plants against harmful organisms, but the physiological mechanisms of their action are just beginning to be studied. In this work, we studied ten strains of bacteria isolated from the tissues of wheat and potatoes. Sequencing of the 16S rRNA gene showed that all isolates belong to the genus Bacillus and to two species, B. subtilis and B. velezensis. The genes for lipopeptide synthetase - surfactin synthetase (Bs_srf ), iturin synthetase (Bs_ituA, Bs_ituB) and fengycin synthetase (Bs_fenD) - were identified in all bacterial isolates using PCR. All strains had high aphicidal activity against the Greenbug aphid (Schizaphis graminum Rond.) due to the synthesis of lipopeptides, which was proven using lipopeptide-rich fractions (LRFs) isolated from the strains. Endophytic lipopeptide-synthesizing strains of Bacillus spp. indirectly affected the viability of aphids, the endurance of plants against aphids and triggered ISR in plants, which manifested itself in the regulation of oxidative metabolism and the accumulation of transcripts of the Pr1, Pr2, Pr3, Pr6 and Pr9 genes due to the synthesis of lipopeptides, which was proven using LRF isolated from three strains: B. subtilis 26D, B. subtilis 11VM, and B. thuringiensis B-6066. We have for the first time demonstrated the aphicidal effect of fengycin and the ability of the fengycin-synthesizing strains and isolates, B. subtilis Ttl2, Bacillus sp. Stl7 and B. thuringiensis B-6066, to regulate components of the pro-/antioxidant system of aphid-infested plants. In addition, this work is the first to demonstrate an elicitor role of fengycin in triggering a systemic resistance to S. graminum in wheat plants. We have discovered new promising strains and isolates of endophytes of the genus Bacillus, which may be included in the composition of new biocontrol agents against aphids. One of the criteria for searching for new bacteria active against phloem-feeding insects can be the presence of lipopeptide synthetase genes in the bacterial genome.202438952706
3640.9690Bacillus amyloliquefaciens SN16-1-Induced Resistance System of the Tomato against Rhizoctonia solani. Tomato (Solanum lycopersicum), as an important economical vegetable, is often infected with Rhizoctonia solani, which results in a substantial reduction in production. Therefore, the molecular mechanism of biocontrol microorganisms assisting tomato to resist pathogens is worth exploring. Here, we use Bacillus amyloliquefaciens SN16-1 as biocontrol bacteria, and employed RNA-Seq technology to study tomato gene and defense-signaling pathways expression. Gene Ontology (GO) analyses showed that an oxidation-reduction process, peptidase regulator activity, and oxidoreductase activity were predominant. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that phenylpropanoid biosynthesis, biosynthesis of unsaturated fatty acids, aldosterone synthesis and secretion, and phototransduction were significantly enriched. SN16-1 activated defenses in the tomato via systemic-acquired resistance (which depends on the salicylic acid signaling pathway), rather than classic induction of systemic resistance. The genes induced by SN16-1 included transcription factors, plant hormones (ethylene, auxin, abscisic acid, and gibberellin), receptor-like kinases, heat shock proteins, and defense proteins. SN16-1 rarely activated pathogenesis-related proteins, but most pathogenesis-related proteins were induced in the presence of the pathogens. In addition, the molecular mechanisms of the response of tomatoes to SN16-1 and R. solani RS520 were significantly different.202135055983
1550.9689Enhanced Bacterial Wilt Resistance in Potato Through Expression of Arabidopsis EFR and Introgression of Quantitative Resistance from Solanum commersonii. Bacterial wilt (BW) caused by Ralstonia solanacearum is responsible for substantial losses in cultivated potato (Solanum tuberosum) crops worldwide. Resistance genes have been identified in wild species; however, introduction of these through classical breeding has achieved only partial resistance, which has been linked to poor agronomic performance. The Arabidopsis thaliana (At) pattern recognition receptor elongation factor-Tu (EF-Tu) receptor (EFR) recognizes the bacterial pathogen-associated molecular pattern EF-Tu (and its derived peptide elf18) to confer anti-bacterial immunity. Previous work has shown that transfer of AtEFR into tomato confers increased resistance to R. solanacearum. Here, we evaluated whether the transgenic expression of AtEFR would similarly increase BW resistance in a commercial potato line (INIA Iporá), as well as in a breeding potato line (09509.6) in which quantitative resistance has been introgressed from the wild potato relative Solanum commersonii. Resistance to R. solanacearum was evaluated by damaged root inoculation under controlled conditions. Both INIA Iporá and 09509.6 potato lines expressing AtEFR showed greater resistance to R. solanacearum, with no detectable bacteria in tubers evaluated by multiplex-PCR and plate counting. Notably, AtEFR expression and the introgression of quantitative resistance from S. commersonii had a significant additive effect in 09509.6-AtEFR lines. These results show that the combination of heterologous expression of AtEFR with quantitative resistance introgressed from wild relatives is a promising strategy to develop BW resistance in potato.201729033958
874860.9688Heterologous Expression of the Constitutive Disease Resistance 2 and 8 Genes from Poncirus trifoliata Restored the Hypersensitive Response and Resistance of Arabidopsis cdr1 Mutant to Bacterial Pathogen Pseudomonas syringae. Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. In the United States, this disease is associated with a phloem-restricted bacterium, Candidatus Liberibacter asiaticus. Commercial citrus cultivars are susceptible to HLB, but Poncirus trifoliata, a close relative of Citrus, is highly tolerant of HLB. Isolating P. trifoliata gene(s) controlling its HLB tolerance followed by expressing the gene(s) in citrus is considered a potential cisgenic approach to engineering citrus for tolerance to HLB. Previous gene expression studies indicated that the constitutive disease resistance (CDR) genes in P. trifoliata (PtCDRs) may play a vital role in its HLB tolerance. This study was designed to use Arabidopsis mutants as a model system to confirm the function of PtCDRs in plant disease resistance. PtCDR2 and PtCDR8 were amplified from P. trifoliata cDNA and transferred into the Arabidopsis cdr1 mutant, whose resident CDR1 gene was disrupted by T-DNA insertion. The PtCDR2 and PtCDR8 transgenic Arabidopsis cdr1 mutant restored its hypersensitive response to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2. The defense marker gene PATHOGENESIS RELATED 1 (PR1) expressed at much higher levels in the PtCDR2 or PtCDR8 transgenic cdr1 mutant than in the non-transgenic cdr1 mutant with or without pathogen infection. Multiplication of Pst DC3000 bacteria in Arabidopsis was inhibited by the expression of PtCDR2 and PtCDR8. Our results showed that PtCDR2 and PtCDR8 were functional in Arabidopsis and played a positive role in disease resistance and demonstrated that Arabidopsis mutants can be a useful alternate system for screening Poncirus genes before making the time-consuming effort to transfer them into citrus, a perennial woody plant that is highly recalcitrant for Agrobacterium or biolistic-mediated transformation.202032629813
875970.9688Genetic and transcriptomic dissection of host defense to Goss's bacterial wilt and leaf blight of maize. Goss's wilt, caused by the Gram-positive actinobacterium Clavibacter nebraskensis, is an important bacterial disease of maize. The molecular and genetic mechanisms of resistance to the bacterium, or, in general, Gram-positive bacteria causing plant diseases, remain poorly understood. Here, we examined the genetic basis of Goss's wilt through differential gene expression, standard genome-wide association mapping (GWAS), extreme phenotype (XP) GWAS using highly resistant (R) and highly susceptible (S) lines, and quantitative trait locus (QTL) mapping using 3 bi-parental populations, identifying 11 disease association loci. Three loci were validated using near-isogenic lines or recombinant inbred lines. Our analysis indicates that Goss's wilt resistance is highly complex and major resistance genes are not commonly present. RNA sequencing of samples separately pooled from R and S lines with or without bacterial inoculation was performed, enabling identification of common and differential gene responses in R and S lines. Based on expression, in both R and S lines, the photosynthesis pathway was silenced upon infection, while stress-responsive pathways and phytohormone pathways, namely, abscisic acid, auxin, ethylene, jasmonate, and gibberellin, were markedly activated. In addition, 65 genes showed differential responses (up- or down-regulated) to infection in R and S lines. Combining genetic mapping and transcriptional data, individual candidate genes conferring Goss's wilt resistance were identified. Collectively, aspects of the genetic architecture of Goss's wilt resistance were revealed, providing foundational data for mechanistic studies.202337652038
3580.9685Gluconacetobacter diazotrophicus Elicits a Sugarcane Defense Response Against a Pathogenic Bacteria Xanthomonas albilineans. A new role for the plant growth-promoting nitrogen-fixing endophytic bacteria Gluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions. Living G.diazotrophicus possess and/or produce elicitor molecules which activate the sugarcane defense response resulting in the plant resistance to X. albilineans, in this particular case controlling the pathogen transmission to emerging agamic shoots. A total of 47 differentially expressed transcript derived fragments (TDFs) were identified by cDNA-AFLP. Transcripts showed significant homologies to genes of the ethylene signaling pathway (26%), proteins regulates by auxins (9%), beta-1,3 Glucanase proteins (6%) and ubiquitin genes (4%), all major signaling mechanisms. Results point toward a form of induction of systemic resistance in sugarcane-G. diazotrophicus interactions which protect the plant against X. albilineans attack.200619516988
4590.9685Vitis vinifera VvNPR1.1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew. Studying grapevine (Vitis vinifera) innate defense mechanisms is a prerequisite to the development of new protection strategies, based on the stimulation of plant signaling pathways to trigger pathogen resistance. Two transcriptional coactivators (VvNPR1.1 and VvNPR1.2) with similarity to Arabidopsis thaliana NPR1 (Non-Expressor of PR genes 1), a well-characterized and key signaling element of the salicylic acid (SA) pathway, were recently isolated in Vitis vinifera. In this study, functional characterization of VvNPR1.1 and VvNPR1.2, including complementation of the Arabidopsis npr1 mutant, revealed that VvNPR1.1 is a functional ortholog of AtNPR1, whereas VvNPR1.2 likely has a different function. Ectopic overexpression of VvNPR1.1 in the Arabidopsis npr1-2 mutant restored plant growth at a high SA concentration, Pathogenesis Related 1 (PR1) gene expression after treatment with SA or bacterial inoculation, and resistance to virulent Pseudomonas syringae pv. maculicola bacteria. Moreover, stable overexpression of VvNPR1.1-GFP in V. vinifera resulted in constitutive nuclear localization of the fusion protein and enhanced PR gene expression in uninfected plants. Furthermore, grapevine plants overexpressing VvNPR1.1-GFP exhibited an enhanced resistance to powdery mildew infection. This work highlights the importance of the conserved SA/NPR1 signaling pathway for resistance to biotrophic pathogens in V. vinifera.201121505863
8195100.9683Comparative proteomics reveals essential mechanisms for osmotolerance in Gluconacetobacter diazotrophicus. Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.202133035671
527110.9682Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128. Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.201930526412
14120.9682Unraveling Pinus massoniana's Defense Mechanisms Against Bursaphelenchus xylophilus Under Aseptic Conditions: A Transcriptomic Analysis. Pine wilt disease (PWD) is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and significantly impacts pine forest ecosystems globally. This study focuses on Pinus massoniana, an important timber and oleoresin resource in China, which is highly susceptible to PWN. However, the defense mechanism of pine trees in response to PWN remains unclear. Addressing the complexities of PWD, influenced by diverse factors such as bacteria, fungi, and environment, we established a reciprocal system between PWN and P. massoniana seedlings under aseptic conditions. Utilizing combined second- and third-generation sequencing technologies, we identified 3,718 differentially expressed genes post PWN infection. Transcript analysis highlighted the activation of defense mechanisms via stilbenes, salicylic acid and jasmonic acid pathways, terpene synthesis, and induction of pathogenesis-related proteins and resistance genes, predominantly at 72 h postinfection. Notably, terpene synthesis pathways, particularly the mevalonate pathway, were crucial in defense, suggesting their significance in P. massoniana's response to PWN. This comprehensive transcriptome profiling offers insights into P. massoniana's intricate defense strategies against PWN under aseptic conditions, laying a foundation for future functional analyses of key resistance genes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.202439283201
22130.9681A plant growth-promoting bacteria Priestia megaterium JR48 induces plant resistance to the crucifer black rot via a salicylic acid-dependent signaling pathway. Xanthomonas campestris pv. campestris (Xcc)-induced black rot is one of the most serious diseases in cruciferous plants. Using beneficial microbes to control this disease is promising. In our preliminary work, we isolated a bacterial strain (JR48) from a vegetable field. Here, we confirmed the plant-growth-promoting (PGP) effects of JR48 in planta, and identified JR48 as a Priestia megaterium strain. We found that JR48 was able to induce plant resistance to Xcc and prime plant defense responses including hydrogen peroxide (H(2)O(2)) accumulation and callose deposition with elevated expression of defense-related genes. Further, JR48 promoted lignin biosynthesis and raised accumulation of frees salicylic acid (SA) as well as expression of pathogenesis-related (PR) genes. Finally, we confirmed that JR48-induced plant resistance and defense responses requires SA signaling pathway. Together, our results revealed that JR48 promotes plant growth and induces plant resistance to the crucifer black rot probably through reinforcing SA accumulation and response, highlighting its potential as a novel biocontrol agent in the future.202236438094
12140.9681A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction. Pine wilt disease (PWD) caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the devastating diseases affecting pine forests worldwide. Although effective control measurements are still missing, induction of resistance could represent a possible eco-friendly alternative. In this study, induced resistance-based in vitro and in vivo screening tests were carried out for selection of bacteria with the ability to suppress PWD. Out of 504 isolated bacteria, Bacillus thuringiensis JCK-1233 was selected for its ability to boost pathogenesis-related 1 (PR1) gene expression, a marker of systemic acquired resistance. Moreover, treatment of pine seedlings with B. thuringiensis JCK-1233 resulted in increased expression of other defense-related genes, and significantly inhibited PWD development under greenhouse conditions. However, B. thuringiensis JCK-1233 showed no direct nematicidal activity against B. xylophilus. To identify the effective compound responsible for the induction of resistance in B. thuringiensis JCK-1233, several diketopiperazines (DPKs) including cyclo-(D-Pro-L-Val), cyclo-(L-Pro-L-Ile), cyclo-(L-Pro-L-Phe), and cyclo-(L-Leu-L-Val) were isolated and tested. Foliar treatment of pine seedlings with Cyclo-(L-Pro-L-Ile) resulted in suppression of PWD severity and increased the expression of defense-related genes similarly to B. thuringiensis JCK-1233 treatment. Interestingly, treatment with B. thuringiensis JCK-1233 or cyclo-(L-Pro-L-Ile) showed moderately enhanced expression of PR-1, PR-2, PR-3, PR-4, PR-5, and PR-9 genes following inoculation with PWN compared to that in the untreated control, indicating that they mitigated the burst of hypersensitive reaction in susceptible pine seedlings. In contrast, they significantly increased the expression levels of PR-6 and PR-10 before PWN inoculation. In conclusion, foliar spraying with either B. thuringiensis JCK-1233 culture suspension or DPKs could induce resistance in pine seedlings, thereby alleviating the serious damage by PWD. Taken together, this study supports aerial spraying with eco-friendly biotic or abiotic agents as a valuable strategy that may mark an epoch for the control of PWD in pine forests.202032849672
9013150.9680Two antimicrobial genes from Aegilops tauschii Cosson identified by the Bacillus subtilis expression system. Antimicrobial genes play an important role as a primary defense mechanism in all multicellular organisms. We chose Bacillus subtilis as a target pathogen indicator and transferred the Aegilops tauschii Cosson cDNA library into B. subtilis cells. Expression of the candidate antimicrobial gene can inhibit B. subtilis cell growth. Using this strategy, we screened six genes that have an internal effect on the indicator bacteria. Then, the secreted proteins were extracted and tested; two genes, AtR100 and AtR472, were found to have strong external antimicrobial activities with broad-spectrum resistance against Xanthomonas oryzae pv. oryzicola, Clavibacter fangii, and Botrytis cinerea. Additionally, thermal stability tests indicated that the antimicrobial activities of both proteins were thermostable. Furthermore, these two proteins exhibited no significant hemolytic activities. To test the feasibility of application at the industrial level, liquid fermentation and spray drying of these two proteins were conducted. Powder dilutions were shown to have significant inhibitory effects on B. cinerea. Fluorescence microscopy and flow cytometry results showed that the purified protein impaired and targeted the cell membranes. This study revealed that these two antimicrobial peptides could potentially be used for replacing antibiotics, which would provide the chance to reduce the emergence of drug resistance.202032770019
8747160.9679An endolysin gene from Candidatus Liberibacter asiaticus confers dual resistance to huanglongbing and citrus canker. The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. citri (Xcc), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from CaLas. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc, according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus, 35S, were integrated into Carrizo citrange via Agrobacterium-mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low CaLas titers and no obvious symptoms while also clearing CaLas from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc, indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of CaLas-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus.202337719271
626170.9679Enterococcus faecalis Adapts to Antimicrobial Conjugated Oligoelectrolytes by Lipid Rearrangement and Differential Expression of Membrane Stress Response Genes. Conjugated oligoelectrolytes (COEs) are emerging antimicrobials with broad spectrum activity against Gram positive and Gram negative bacteria as well as fungi. Our previous in vitro evolution studies using Enterococcus faecalis grown in the presence of two related COEs (COE1-3C and COE1-3Py) led to the emergence of mutants (changes in liaF and liaR) with a moderate 4- to16-fold increased resistance to COEs. The contribution of liaF and liaR mutations to COE resistance was confirmed by complementation of the mutants, which restored sensitivity to COEs. To better understand the cellular target of COEs, and the mechanism of resistance to COEs, transcriptional changes associated with resistance in the evolved mutants were investigated in this study. The differentially transcribed genes encoded membrane transporters, in addition to proteins associated with cell envelope synthesis and stress responses. Genes encoding membrane transport proteins from the ATP binding cassette superfamily were the most significantly induced or repressed in COE tolerant mutants compared to the wild type when exposed to COEs. Additionally, differences in the membrane localization of a lipophilic dye in E. faecalis exposed to COEs suggested that resistance was associated with lipid rearrangement in the cell membrane. The membrane adaptation to COEs in EFC3C and EFC3Py resulted in an improved tolerance to bile salt and sodium chloride stress. Overall, this study showed that bacterial cell membranes are the primary target of COEs and that E. faecalis adapts to membrane interacting COE molecules by both lipid rearrangement and changes in membrane transporter activity. The level of resistance to COEs suggests that E. faecalis does not have a specific response pathway to elicit resistance against these molecules and this is supported by the rather broad and diverse suite of genes that are induced upon COE exposure as well as cross-resistance to membrane perturbing stressors.202032117172
21180.9679miR159a modulates poplar resistance against different fungi and bacteria. Trees are inevitably attacked by different kinds of pathogens in their life. However, little is known about the regulatory factors in poplar response to different pathogen infections. MicroRNA159 (miR159) is a highly conserved microRNA (miRNA) in plants and regulates plant development and stress responses. Here, transgenic poplar overexpressing pto-miR159a (OX-159) showed antagonistic regulation mode to poplar stem disease caused by fungi Cytospora chrysosperma and bacteria Lonsdalea populi. OX-159 lines exhibited a higher susceptibility after inoculation with bacterium L. populi, whereas enhanced disease resistance to necrotrophic fungi C. chrysosperma compared with wild-type (WT) poplars. Intriguingly, further disease assay found that OX159 line rendered the poplar susceptible to hemi-biotrophic fungi Colletotrichum gloeosporioide, exhibiting larger necrosis and lower ROS accumulation than WT lines. Transcriptome analyses revealed that more down-regulated differentially expressed genes with disease-resistant domains in OX-159 line compared with WT line. Moreover, the central mediator NPR1 of salicylic acid (SA) pathway showed a decrease in expression level, while jasmonic acid/ethylene (JA/ET) signal pathway marker genes ERF, as well as PR3, MPK3, and MPK6 genes showed an increase level in OX159-2 and OX159-5 compared with WT lines. Further spatio-temporal expression analysis revealed JA/ET signaling was involved in the dynamic response process to C. gloeosporioides in WT and OX159 lines. These results demonstrate that overexpression of pto-miR159a resulted in the crosstalk changes of the downstream hub genes, thereby controlling the disease resistance of poplars, which provides clues for understanding pto-miR159a role in coordinating poplar-pathogen interactions.202337494825
57190.9679Functional analysis of NtMPK2 uncovers its positive role in response to Pseudomonas syringae pv. tomato DC3000 in tobacco. Mitogen-activated protein kinase cascades are highly conserved signaling modules downstream of receptors/sensors and play pivotal roles in signaling plant defense against pathogen attack. Extensive studies on Arabidopsis MPK4 have implicated that the MAP kinase is involved in multilayered plant defense pathways. In this study, we identified tobacco NtMPK2 as an ortholog of AtMPK4. Transgenic tobacco overexpressing NtMPK2 markedly enhances resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) virulent and avirulent strains. Transcriptome analysis of NtMPK2-dependent genes shows that possibly the basal resistance system is activated by NtMPK2 overexpression. In addition to NtMPK2-mediated resistance, multiple pathways are involved in response to the avirulent bacteria based on analysis of Pst-responding genes, including SA and ET pathways. Notably, it is possible that biosynthesis of antibacterial compounds is responsible for inhibition of Pst DC3000 avirulent strain when programmed cell death processes in the host. Our results uncover that NtMPK2 positively regulate tobacco defense response to Pst DC3000 and improve our understanding of plant molecular defense mechanism.201626482478