Cell Wall-degrading Enzymes Research Articles

Hormonal and proteomic analyses of southern blight disease caused by Athelia rolfsii and root chitosan priming on Cannabis sativa in an in vitro hydroponic system.

Southern blight disease, caused by the fungal pathogen Athelia rolfsii, suppresses plant growth and reduces product yield in Cannabis sativa agriculture. Mechanisms of pathology of this soil-borne disease remain poorly understood, with disease management strategies reliant upon broad-spectrum antifungal use. Exposure to chitosan, a natural elicitor, has been proposed as an alternative method to control diverse fungal diseases in an eco-friendly manner. In this study, C.sativa plants were grown in the Root-TRAPR system, a transparent hydroponic growth device, where plant roots were primed with .2% colloidal chitosan prior to A.rolfsii inoculation. Both chitosan-primed and unprimed inoculated plants displayed classical symptoms of wilting and yellowish leaves, indicating successful infection. Non-primed infected plants showed increased shoot defense responses with doubling of peroxidase and chitinase activities. The levels of growth and defense hormones including auxin, cytokinin, and jasmonic acid were increased 2-5-fold. In chitosan-primed infected plants, shoot peroxidase activity and phytohormone levels were decreased 1.5-4-fold relative to the unprimed infected plants. When compared with shoots, roots were less impacted by A.rolfsii infection, but the pathogen secreted cell wall-degrading enzymes into the root-growth solution. Chitosan priming inhibited root growth, with root lengths of chitosan-primed plants approximately 65% shorter than the control, but activated root defense responses, with root peroxidase activity increased 2.7-fold along with increased secretion of defense proteins. The results suggest that chitosan could be an alternative platform to manage southern blight disease in C.sativa cultivation; however, further optimization is required to maximize effectiveness of chitosan.

The Adenylate Cyclase-Encoding Gene crac Is Involved in Clonostachys rosea Mycoparasitism.

Clonostachys rosea is an excellent biocontrol fungus against numerous fungal plant pathogens. The cAMP signaling pathway is a crucial signal transduction pathway in fungi. To date, the role of the cAMP signaling pathway in C. rosea mycoparasitism remains unknown. An adenylate cyclase-encoding gene, crac (an important component of the cAMP signaling pathway), was previously screened from C. rosea 67-1, and its expression level was dramatically upregulated during the C. rosea mycoparasitization of the sclerotia of Sclerotinia sclerotiorum. In this study, the function of crac in C. rosea mycoparasitism was explored through gene knockout and complementation. The obtained results show that the deletion of crac influenced the growth rate and colony morphology of C. rosea, as well as the tolerance to NaCl and H2O2 stress. The mycoparasitic effects on the sclerotia of S. sclerotiorum and the biocontrol capacity on soybean Sclerotinia stem rot in ∆crac-6 and ∆crac-13 were both attenuated compared with that of the wild-type strain and complementation transformants. To understand the regulatory mechanism of crac during C. rosea mycoparasitism, transcriptomic analysis was conducted between the wild-type strain and knockout mutant. A number of biocontrol-related genes, including genes encoding cell wall-degrading enzymes and transporters, were significantly differentially expressed during C. rosea mycoparasitism, suggesting that crac may be involved in C. rosea mycoparasitism by regulating the expression of these DEGs. These findings provide insight for further exploring the molecular mechanism of C. rosea mycoparasitism.

Low Molecular Weight and High Deacetylation Degree Chitosan Batch Alleviates Pathogenesis, Toxin Accumulation, and Fusarium Gene Regulation in Barley Leaf Pathosystem.

Fusarium graminearum is a cosmopolitan fungal pathogen that destroys cereal production, in terms of loss of yield and grain contamination with mycotoxins, worldwide. Chitosan is a natural biopolymer abundant in the environment with proven antifungal properties that also acts as a plant immunity elicitor. Despite a number of articles, there is a lack of systematic comparison of antifungal activity of diverse batches of chitosan. The current study aimed to test the inhibitory effects of a collection of diverse chitosan samples on the growth and production of F. graminearum toxins, validated by changes in the Fusarium transcriptome. Experiments included testing antifungal activity of different chitosan samples, the application of the best performing one in vitro to investigate the impact on F. graminearum growth, followed by analyzing its effect on Fusarium toxins accumulation, and Fusarium transcriptomics in the barley leaf pathosystem. Confirmatory antifungal assays revealed that CS_10, a specific batch of chitosan, retarded Fusarium growth with an application concentration of 200 ppm, significantly reducing toxin synthesis and disease symptoms in Fusarium-inoculated barley leaves. RNA-Seq analysis of F. graminearum in barley leaf pathosystem exposed to CS_10 showed a list of differentially expressed genes involved in redox balance, cell respiration, nutrient transport, cell wall degradation enzymes, ergosterol biosynthesis, and trichothecenes production. The genes functioning in these essential pathways are discussed and assigned as critical checkpoints to control Fusarium infections. The results suggest some important molecular targets in F. graminearum that may be suitable in gene-specific targeting or transgene-free methods, such as spray-induced gene silencing during host-pathogen interactions.

Diversity of transgenes in sustainable management of insect pests.

Insecticidal transgenes, when incorporated and expressed in plants, confer resistance against insects by producing several products having insecticidal properties. Protease inhibitors, lectins, amylase inhibitors, and chitinase genes are associated with the natural defenses developed by plants to counter insect attacks. Several toxin genes are also derived from spiders and scorpions for protection against insects. Bacillus thuringiensis Berliner is a microbial source of insecticidal toxins. Several methods have facilitated the large-scale production of transgenic plants. Bt-derived cry, cyt, vip, and sip genes, plant-derived genes such as lectins, protease inhibitors, and alpha-amylase inhibitors, insect cell wall-degrading enzymes like chitinase and some proteins like arcelins, plant defensins, and ribosome-inactivating proteins have been successfully utilized to impart resistance to insects. Besides, transgenic plants expressing double-stranded RNA have been developed with enhanced resistance. However, the long-term effects of transgenes on insect resistance, the environment, and human health must be thoroughly investigated before they are made available for commercial planting. In this chapter, the present status, prospects, and future scope of transgenes for insect pest management have been summarized and discussed.

High-throughput RNA sequencing reveals differences between the transcriptomes of the five spore forms of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen

The devastating wheat stripe (yellow) rust pathogen, Puccinia striiformis f. sp. tritici (Pst), is a macrocyclic and heteroecious fungus. Pst produces urediniospores and teliospores on its primary host, wheat, and pycniospores and aeciospores are produced on its alternate hosts, barberry (Berberis spp.) or mahonia (Mahonia spp.). Basidiospores are developed from teliospores and infect alternate hosts. These five spore forms play distinct roles in Pst infection, disease development, and fungal survival, etc. However, the specific genes and mechanisms underlying these functional differences are largely unknown. In this study, we performed, for the first time in rust fungi, the deep RNA sequencing to examine the transcriptomic shift among all five Pst spore forms. Among a total of 29,591 identified transcripts, 951 were specifically expressed in basidiospores, whereas 920, 761, 266, and 110 were specific for teliospores, pycniospores, aeciospores, and urediniospores, respectively. Additionally, transcriptomes of sexual spores, namely pycniospores and basidiospores, showed significant differences from those of asexual spores (urediniospores, teliospores, and aeciospores), and transcriptomes of urediniospores and aeciospores were more similar to each other than to the three other spore forms. Especially, the basidiospores and pycniospores which infected the berberis shows wide differences in the cell wall degrading-enzymes and mating and pheromone response genes. Besides, we also found that there are 6234 differential expressed genes between the urediniospores and pycniospores, while only have 3 genes have alternative splicing enents, suggesting that differential genes expression may make more contribution than AS. This comprehensive transcriptome profiling can substantially improve our understanding of the developmental biology of the wheat stripe rust fungus.

A seed-endophytic bacterium NEAU-242-2: Isolation, identification, and potential as a biocontrol agent against Bipolaris sorokiniana

Currently, common root rot of wheat has caused significant yield losses worldwide. Biological control is an important method to control common root rot of wheat. An endophytic bacterium, strain NEAU-242-2 was isolated from wheat seeds, which showed the strongest inhibitory activity against Bipolaris sorokiniana (84.2%) and also had strong broad-spectrum antifungal activity, with the inhibition rates over 61.2% against all phytopathogens used in this study except for Rhizoctonia solani (44.5%), and was identified as Bacillus velezensis by 16S rRNA gene sequence and whole genome sequence and average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values. Pot experiments showed that strain NEAU-242-2 exhibited significant biocontrol effect (80.61 ± 1.22%) on common root rot of wheat in greenhouse. Furthermore, the sterile culture filtrate of strain NEAU-242-2 had strong inhibitory activity (86.96 ± 3.02%) against B. sorokiniana and the main antifungal substance was preliminarily identified as lipopeptides. The crude lipopeptide extracted from strain NEAU-242-2 showed strong antifungal activity (61.45 ± 3.45%) against B. sorokiniana with the IC50 of 25.93 µg/mL, and could cause mycelial wrinkle, fracture, and dehydrated malformation, destroy the structure of cell, and lead to cellular malformation and the disappearance of most membrane-like structures. Besides that, strain NEAU-242-2 could also conspicuously promote wheat seed germination and seedling growth. The fungistasis and plant growth-promoting activity of strain NEAU-242-2 were found to be associated with the production of IAA (19.4 ± 0.6 µg/mL), siderophores, nitrogenases, cell wall-degrading enzymes (proteases and cellulases), and other active secondary metabolites. Additionally, a large number of genes related to antifungal and plant growth-promoting traits were also identified in the whole genome of strain NEAU-242-2. These results indicated that strain NEAU-242-2 had the potential as a biocontrol agent for B. sorokiniana.

Diversity and Biocontrol Potential of Endophytic Fungi and Bacteria Associated with Healthy Welsh Onion Leaves in Taiwan.

Foliar diseases caused by Stemphylium and Colletotrichum species are among the major biotic factors limiting Welsh onion production in Taiwan. Owing to concerns about the environment and the development of pathogen resistance to existing fungicides, biological control using endophytes is emerging as an eco-friendly alternative to chemical control. The aim of the present study was to isolate endophytes from healthy Welsh onion leaves and investigate their antagonistic potential against the major phytopathogenic fungi associated with Welsh onion plants in Taiwan. A total of 109 bacterial and 31 fungal strains were isolated from healthy Welsh onion leaves and assigned to 16 bacterial and nine fungal genera using morphological and molecular characterization based on DNA sequence data obtained from nuclear internal transcribed spacer (nrITS) (fungi) and 16S rRNA (bacteria). Evaluation of these endophytic isolates for biocontrol activity against leaf blight pathogens Colletotrichum spaethianum strain SX15-2 and Stemphylium vesicarium strain SX20-2 by dual culture assay and greenhouse experiments resulted in the identification of two bacterial isolates (GFB08 and LFB28) and two fungal isolates (GFF06 and GFF08) as promising antagonists to leaf blight pathogens. Among the four selected isolates, Bacillus strain GFB08 exhibited the highest disease control in the greenhouse study. Therefore, Bacillus strain GFB08 was further evaluated to understand the mechanism underlying its biocontrol efficacy. A phylogenetic analysis based on six genes identified Bacillus strain GFB08 as B. velezensis. The presence of antimicrobial peptide genes (baer, bamC, bmyB, dfnA, fenD, ituC, mlna, and srfAA) and the secretion of several cell wall degrading enzymes (CWDEs), including cellulase and protease, confirmed the antifungal nature of B. velezensis strain GFB08. Leaf blight disease suppression by preventive and curative assays indicated that B. velezensis strain GFB08 has preventive efficacy on C. spaethianum strain SX15-2 and both preventive and curative efficacy on S. vesicarium strain SX20-2. Overall, the current study revealed that healthy Welsh onion leaves harbour diverse bacterial and fungal endophytes, among which the endophytic bacterial strain, B. velezensis strain GFB08, could potentially be used as a biocontrol agent to manage the leaf blight diseases of Welsh onion in Taiwan.

Electrostatic Atomized Water Particles Induce Disease Resistance in Muskmelon (Cucumis melo L.) against Postharvest Fruit Rot Caused by Fusarium incarnatum.

The postharvest quality of muskmelon can be affected by fruit rot caused by the fungus Fusarium incarnatum, resulting in loss of quality. The utilization of electrostatic atomized water particles (EAWPs) in agriculture applications has been shown to induce disease resistance in plants. Therefore, in this study, we determined the effect of electrostatic atomized water particles (EAWPs) on the disease resistance of muskmelon fruits against postharvest fruit rot caused by F. incarnatum. EAWPs were applied to muskmelon fruits for 0, 30, 60, and 90 min. EAWP-treated muskmelon fruits were inoculated with F. incarnatum, and disease progress was measured. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of the chitinase (CmCHI) and β-1,3-glucanase (CmGLU) genes of Cucumis melo (muskmelon) was performed for EAWP-treated and -untreated muskmelon fruits. The activities of cell-wall-degrading enzymes (CWDEs), chitinase, and β-1,3-glucanase were also assayed in EAWP-treated and -untreated muskmelon fruits. The results showed that disease progress was limited by EAWP treatment for 30 min prior to pathogen inoculation. Muskmelon fruits treated with EAWPs for 30 min showed an upregulation of CWDE genes, CmCHI and CmGLU, as observed by qRT-PCR, leading to high chitinase and β-1,3-glucanase activities, as observed through enzyme assays. The results of SEM microscopy revealed that the effect of the crude enzymes of EAWP-treated muskmelon caused morphological changes in F. incarnatum mycelia. Furthermore, treatment with EAWPs preserved postharvest quality in muskmelon, including with regard to texture stiffness and total chlorophyll contents, compared to untreated muskmelon. These results demonstrate that the pretreatment of muskmelon with EAWPs suppresses the development of F. incarnatum in the early stage of infection by regulating gene expression of CWDEs and elevating the activities of CWDEs, while also maintaining postharvest muskmelon quality.

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus

Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other’s recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation.

Effects of Hard Water Boiling on Chalky Rice in Terms of Texture Improvement and Ca Fortification.

In the present paper, we investigated the characteristics of chalky rice grains generated by ripening under high temperature and compared them with whole grains. We evaluated 14 unpolished Japonica rice grains harvested in Japan in 2021, and these samples (original grains) were divided into two groups (a whole grain group and a chalky grain one). We found that not only activities of endogenous amylase and proteinase, but also cell wall-degrading enzymes, such as xylanase and cellulase, changed markedly between chalky grains and whole grains. Using rice grains blended with 30% of chalky grains as the material, we compared the sugar and mineral contents and textural properties of the rice grains soaked and boiled in either ordinary water or hard water, such as Evian or Contrex. It was shown that xylanase, in addition to amylase and proteinase, may play an important role in changing the texture of the boiled chalky rice grains. For the sake of preventing the above-mentioned deterioration in the texture of boiled grains of chalky rice, we tried to use hard water, such as Evian or Contrex, to soak and cook the chalky rice grains. It was shown that the hard water was useful for the prevention of texture deterioration of the boiled rice grains due to inhibition of the activities of endogenous hydrolytic enzymes, such as α-amylase, β-amylase, proteinase, and xylanase. Furthermore, we found that the hard water was useful in increasing the calcium absorption through the meal by 2.6 to 16.5 times.

Postharvest Calcium Chloride Treatment Strengthens Cell Wall Structure to Maintain Litchi Fruit Quality.

Litchi (Litchi chinensis Sonn.) fruit deterioration occurs rapidly after harvest and is characterized by pericarp browning, pulp softening, and decay. In this study, we found that calcium chloride (CaCl2) treatment (5 g L-1 CaCl2 solution vacuum infiltration for 5 min) affected the cell wall component contents and cell wall-degrading enzyme activities of litchi fruit during storage at room temperature. CaCl2 treatment significantly increased the contents of Ca2+ and cellulose, while it decreased the water-soluble pectin content, and the activities of polygalacturonase, β-galactosidase, and cellulase in the litchi pericarp. Meanwhile, the treatment resulted in significantly increased contents of Ca2+, water-soluble pectin, ionic-soluble pectin, covalent-soluble pectin and hemicellulose, and upregulated activities of pectinesterase and β-galactosidase, while significantly decreasing the activities of polygalacturonase and cellulase in litchi pulp. The above results indicate that CaCl2 treatment strengthened the cell wall structure of litchi fruit. More importantly, the enzymatic browning of the pericarp, softening of the pulp, and disease incidence were delayed. The treatment had a more pronounced effect on the pericarp than on the pulp. We consider CaCl2 treatment to be a safe and effective treatment for maintaining the postharvest quality of litchi fruit.

Effect of enzyme-response Mag@CMCS@CPSG film on rice sheath blight disease (caused by Rhizoctonia solani) control and rice quality

To find a precise, efficient, and safe method to control rice sheath blight disease (caused by Rhizoctonia solani), this study used the natural product magnolol as the fungicide. Based on the characteristics of cell wall-degrading enzymes (CWDEs) released by R. solani during the early stages of infection, a Mag@CMCS@CPSG (magnolol@carboxymethyl chitosan@carboxymethyl cellulose, pectin, sodium alginate, and glycerol) film was constructed to control the release of magnolol in response to CWDEs. The Mag@CMCS@CPSG film increased magnolol toxicity against R. solani, decreased EC50 from 7.33 μg/mL to 4.37 μg/mL, and effectively blocked the invasion of mycelia. Under the stimulation of CWDEs released during R. solani infection, the cumulative release of magnolol by the Mag@CMCS@CPSG film reached 75.62% after 96 h, and the disease index of rice treated with Mag@CMCS@CPSG film significantly decreased. The maximum field control effect of Mag@CMCS@CPSG film on rice sheath blight disease was 72.47%, which was not significantly different from that of the commercial preparation jinggangmycin; however, the holding period was better. The Mag@CMCS@CPSG film reduced the loss of rice yield and quality caused by infection with R. solani and did not cause phytotoxicity or residue. In conclusion, the Mag@CMCS@CPSG film can achieve accurate and green control of rice sheath blight.

Intraspecific variability in growth and in vitro production of plant cell wall-degrading enzymes among argentinean isolates of Colletotrichum graminicola, a maize pathogen

Background and aims: Colletotrichum graminicola (Glomerellaceae, Glomerellales), the causal agent of maize (Zea mays) anthracnose, as many other fungal pathogens, relies on its battery of cell wall degrading enzymes (CWDEs) to make its way through the cell walls of the host, and thus the production of these enzymes is considered a virulence factor. Since this disease is relatively new in Argentina, little is known about the capacity of CWDEs production in native isolates. The aim of this work was to investigate if there is intraspecific variability in growth and in vitro production of several extracellular CWDEs among Argentinean fungal isolates of C. graminicola. M&M: Eight isolates of C. graminicola (obtained from symptomatic maize plants growing in different geographical locations) were tested in vitro to evaluate growth capacity and polygalacturonase, polymethylgalacturonase, β-glucosidase and laccase production, using two different liquid culture media. Results: Polygalacturonase, polymethylgalacturonase and β-glucosidase production greatly varied among isolates and culture media. Laccase activity was detected only in three isolates. Utmost enzymatic titres attained were respectively 250, 280, 45 and 63 U/l. The observed intraspecific variability in CWDEs in vitro production is consistent with the high variability found at genetic level when assessing ISSR markers. Conclusions: The isolates of C. graminicola evaluated showed notable differences in CWDEs production, not associated with a differential growth. This indicates a large intraspecific variability, which might be considered when choosing a method to deal with this pathogen.

Antifungal Activities of Fluorinated Pyrazole Aldehydes on Phytopathogenic Fungi, and Their Effect on Entomopathogenic Nematodes, and Soil-Beneficial Bacteria.

Fluoro-substituted pyrazoles have a wide range of biological activities, such as antibacterial, antiviral, and antifungal activities. The aim of this study was to evaluate the antifungal activities of fluorinated 4,5-dihydro-1H-pyrazole derivatives on four phytopathogenic fungi: Sclerotinia sclerotiorum, Macrophomina phaseolina, Fusarium oxysporum f. sp. lycopersici, and F. culmorum. Moreover, they were tested on two soil beneficial bacteria-Bacillus mycoides and Bradyrhizobium japonicum-as well as two entomopathogenic nematodes (EPNs)-Heterorhabditis bacteriophora and Steinernema feltiae. The molecular docking was performed on the three enzymes responsible for fungal growth, the three plant cell wall-degrading enzymes, and acetylcholinesterase (AChE). The most active compounds against fungi S. sclerotiorum were 2-chlorophenyl derivative (H9) (43.07% of inhibition) and 2,5-dimethoxyphenyl derivative (H7) (42.23% of inhibition), as well as H9 against F. culmorum (46.75% of inhibition). Compounds were shown to be safe for beneficial soil bacteria and nematodes, except for compound H9 on EPN H. bacteriophora (18.75% mortality), which also showed the strongest inhibition against AChE (79.50% of inhibition). The molecular docking study revealed that antifungal activity is possible through the inhibition of proteinase K, and nematicidal activity is possible through the inhibition of AChE. The fluorinated pyrazole aldehydes are promising components of future plant protection products that could be environmentally and toxicologically acceptable.

Novel Transcription Factor CXRD Regulates Cellulase and Xylanase Biosynthesis in Penicillium oxalicum under Solid-State Fermentation.

Penicillium oxalicum produces an integrated, extracellular cellulase and xylanase system, strictly regulated by several transcription factors. However, the understanding of the regulatory mechanism of cellulase and xylanase biosynthesis in P. oxalicum is limited, particularly under solid-state fermentation (SSF) conditions. In our study, deletion of a novel gene, cxrD (cellulolytic and xylanolytic regulator D), resulted in 49.3 to 2,230% enhanced production of cellulase and xylanase, except for 75.0% less xylanase at 2 days, compared with the P. oxalicum parental strain, when cultured on solid medium containing wheat bran plus rice straw for 2 to 4 days after transfer from glucose. In addition, the deletion of cxrD delayed conidiospore formation, leading to 45.1 to 81.8% reduced asexual spore production and altered mycelial accumulation to various extents. Comparative transcriptomics and real-time quantitative reverse transcription-PCR found that CXRD dynamically regulated the expression of major cellulase and xylanase genes and conidiation-regulatory gene brlA under SSF. In vitro electrophoretic mobility shift assays demonstrated that CXRD bound to the promoter regions of these genes. The core DNA sequence 5'-CYGTSW-3' was identified to be specifically bound by CXRD. These findings will contribute to understanding the molecular mechanism of negative regulation of fungal cellulase and xylanase biosynthesis under SSF. IMPORTANCE Application of plant cell wall-degrading enzymes (CWDEs) as catalysts in biorefining of lignocellulosic biomass into bioproducts and biofuels reduces both chemical waste production and carbon footprint. The filamentous fungus Penicillium oxalicum can secrete integrated CWDEs, with potential for industrial application. Solid-state fermentation (SSF), simulating the natural habitat of soil fungi, such as P. oxalicum, is used for CWDE production, but a limited understanding of CWDE biosynthesis hampers the improvement of CWDE yields through synthetic biology. Here, we identified a novel transcription factor CXRD, which negatively regulates the biosynthesis of cellulase and xylanase in P. oxalicum under SSF, providing a potential target for genetic engineering to improve CWDE production.

Comparative Characterization of Virulent and Less-Virulent Lasiodiplodia theobromae Isolates.

Lasiodiplodia theobromae attacks over 500 plant species and is an important pathogen of tropical and subtropical fruit. Due to global warming and climate change, the incidence of disease associated with L. theobromae is rising. Virulence tests performed on avocado and mango branches and fruit showed a large diversity of virulence of different L. theobromae isolates. Genome sequencing was performed for two L. theobromae isolates, representing more virulent (Avo62) and less-virulent (Man7) strains, to determine the cause of their variation. Comparative genomics, including orthologous and single-nucleotide polymorphism (SNP) analyses, identified SNPs in the less-virulent strain in genes related to secreted cell wall-degrading enzymes, stress, transporters, sucrose, and proline metabolism, genes in secondary metabolic clusters, effectors, genes involved in the cell cycle, and genes belonging to transcription factors that may contribute to the virulence of L. theobromae. Moreover, carbohydrate-active enzyme analysis revealed a minor increase in gene counts of cutinases and pectinases and the absence of a few glycoside hydrolases in the less-virulent isolate. Changes in gene-copy numbers might explain the morphological differences found in the in-vitro experiments. The more virulent Avo62 grew faster on glucose, sucrose, or starch as a single carbon source. It also grew faster under stress conditions, such as osmotic stress, alkaline pH, and relatively high temperature. Furthermore, the more virulent isolate secreted more ammonia than the less-virulent one both in vitro and in vivo. These study results describe genome-based variability related to L. theobromae virulence, which might prove useful for the mitigation of postharvest stem-end rot. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Variable Tandem Glycine-Rich Repeats Contribute to Cell Death-Inducing Activity of a Glycosylphosphatidylinositol-Anchored Cell Wall Protein That Is Associated with the Pathogenicity of Sclerotinia sclerotiorum.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in eukaryotes. GPI-anchored proteins are widely distributed in fungal plant pathogens, but the specific roles of the GPI-anchored proteins in the pathogenicity of Sclerotinia sclerotiorum, a devastating necrotrophic plant pathogen with a worldwide distribution, remain largely unknown. This research addresses SsGSR1, which encodes an S. sclerotiorum glycine- and serine-rich protein named SsGsr1 with an N-terminal secretory signal and a C-terminal GPI-anchor signal. SsGsr1 is located at the cell wall of hyphae, and deletion of SsGSR1 leads to abnormal cell wall architecture and impaired cell wall integrity of hyphae. The transcription levels of SsGSR1 were maximal in the initial stage of infection, and SsGSR1-deletion strains showed impaired virulence in multiple hosts, indicating that SsGSR1 is critical for the pathogenicity. Interestingly, SsGsr1 targeted the apoplast of host plants to induce cell death that relies on the glycine-rich 11-amino-acid repeats arranged in tandem. The homologs of SsGsr1 in Sclerotinia, Botrytis, and Monilinia species contain fewer repeat units and have lost their cell death activity. Moreover, allelic variants of SsGSR1 exist in field isolates of S. sclerotiorum from rapeseed, and one of the variants lacking one repeat unit results in a protein that exhibits loss of function relative to the cell death-inducing activity and the virulence of S. sclerotiorum. Taken together, our results demonstrate that a variation in tandem repeats provides the functional diversity of GPI-anchored cell wall protein that, in S. sclerotiorum and other necrotrophic pathogens, allows successful colonization of the host plants. IMPORTANCE Sclerotinia sclerotiorum is an economically important necrotrophic plant pathogen and mainly applies cell wall-degrading enzymes and oxalic acid to kill plant cells before colonization. In this research, we characterized a glycosylphosphatidylinositol (GPI)-anchored cell wall protein named SsGsr1, which is critical for the cell wall architecture and the pathogenicity of S. sclerotiorum. Additionally, SsGsr1 induces rapid cell death of host plants that is dependent on glycine-rich tandem repeats. Interestingly, the number of repeat units varies among homologs and alleles of SsGsr1, and such a variation creates alterations in the cell death-inducing activity and the role in pathogenicity. This work advances our understanding of the variation of tandem repeats in accelerating the evolution of a GPI-anchored cell wall protein associated with the pathogenicity of necrotrophic fungal pathogens and prepares the way toward a fuller understanding of the interaction between S. sclerotiorum and host plants.

Transcriptomic and metabolomic analyses reveal molecular mechanisms associated with the natural abscission of blue honeysuckle (Lonicera caerulea L.) ripe fruits

Blue honeysuckle (Lonicera caerulea L.) is rich in phenolic compounds and has an extremely high nutritional value. Fruit abscission in the ripe period significantly impacts production and economic benefits. However, the mechanism associated with the abscission of blue honeysuckle fruit remains largely unknown. The easy-abscission cultivar ‘HSY’ and the hard-abscission cultivar ‘Berel’ were selected as plant materials. Anatomical changes of the ‘HSY’ fruit abscission zone (FAZ) during the abscission mainly included cell expansion, detachment, and collapse. Active changes in cell wall-degrading enzyme activity between 39 days postanthesis (DPA) and 55 DPA in ‘HSY’ FAZ, but not in ‘Berel’, suggest a critical role for cell-wall-degrading enzymes in regulating abscission. Transcriptome and metabolome analyses revealed that the genes and metabolites responding to abscission mainly act on pathways such as plant hormone signal transduction, starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis. The regulatory pathways of fruit abscission are mainly summarized into two parts: phytohormone synthesis and signal transduction, FAZ cell wall metabolism. In this study, 46 key genes related to plant hormone response, 45 key genes involved in FAZ cell wall metabolism, and 73 transcription factors were screened. Quantitative real-time PCR (qRT-PCR) assessed the expression pattern of 12 selected candidate genes, demonstrating the accuracy of the transcriptome data and elucidating the expression patterns of key candidate genes during growth and development. This study will provide an essential resource for understanding the molecular regulatory mechanism of fruit abscission in the blue honeysuckle.

Genome sequencing provides insights into the evolution of gene families encoding plant cell wall-degrading enzymes in longhorned beetles.

With more than 36,000 species, the longhorned beetles (family Cerambycidae) are a mega-diverse lineage of mostly xylophagous insects, all of which are represented by the sole sequenced genome of the Asian longhorned beetle (Anoplophora glabripennis; Lamiinae). Their successful radiation has been linked to their ability to degrade plant cell wall components using a range of so-called plant cell wall-degrading enzymes (PCWDEs). Our previous analysis of larval gut transcriptomes demonstrated that cerambycid beetles horizontally acquired genes encoding PCWDEs from various microbial donors; these genes evolved through multiple duplication events to form gene families. To gain further insights into the evolution of these gene families during the Cerambycidae radiation, we assembled draft genomes for four beetle species belonging to three subfamilies using long-read nanopore sequencing. All the PCWDE-encoding genes we annotated from the corresponding larval gut transcriptomes were present in these draft genomes. We confirmed that the newly discovered horizontally acquired glycoside hydrolase family 7 (GH7), subfamily 26 of GH43 (GH43_26), and GH53 (all of which are absent from the A. glabripennis genome) were indeed encoded by these beetles' genome. Most of the PCWDE-encoding genes of bacterial origin gained introns after their transfer into the beetle genome. Altogether, we show that draft genome assemblies generated from nanopore long-reads offer meaningful information to the study of the evolution of gene families in insects. We anticipate that our data will support studies aiming to better understand the biology of the Cerambycidae and other beetles in general.

Comparative Functional Genome Analysis Reveals the Habitat Adaptation and Biocontrol Characteristics of Plant Growth-Promoting Bacteria in NCBI Databases.

Plant growth-promoting bacteria (PGPB) are a group of beneficial microorganisms that include 60 bacterial genera, such as Bacillus, Pseudomonas, and Burkholderia, which widely colonize plant leaves and soil, promote plant growth, and/or inhibit pathogen infection. However, the genetic factors underpinning adaptation of PGPB to plant leaves and soil remain poorly understood. In this study, we performed a comparative functional genome analysis approach to investigate the functional genes of 195 leaf-associated (LA) and 283 soil-associated (SA) PGPB strains and their roles in adapting to their environment, using 95 strains from other-associated (OA) environmental habitats with growth-promoting or antimicrobial functions as negative controls. Comparison analysis of the enrichment of nonredundant (NR) protein sequence databases showed that cytochrome P450, DNA repair, and motor chemotaxis genes were significantly enriched in LA PGPB strains related to environmental adaptation, while cell wall-degrading enzymes, TetR transcriptional regulatory factors, and sporulation-related genes were highly enriched in SA PGPB strains. Additionally, analysis of carbohydrate-active enzymes demonstrated that glycosyltransferases (GTs) and glycoside hydrolases (GHs) were abundant families in all PGPB strains, which is in favor of plant growth, and enriched in SA PGPB strains. Except for most Bacillus strains, SA PGPB genomes contained significantly more secondary metabolism clusters than LA PGPB. Most LA PGPB contained hormone biosynthesis genes, which may contribute to plant growth promotion, while SA PGPB harbored numerous carbohydrate and antibiotic metabolism genes. In summary, this study further deepens our understanding of the habitat adaptation and biocontrol characteristics of LA and SA PGPB strains. IMPORTANCE Plant growth-promoting bacteria (PGPB) are essential for the effectiveness of biocontrol agents in plant phyllosphere and rhizosphere. However, little is known about the ecological adaptation of PGPB to different habitats. In this study, comparative functional genome analysis of leaf-associated (LA), soil-associated (SA), and other-associated (OA) PGPB strains was performed. We found that genes related to the metabolism of hormones were enriched in LA PGPB. Carbohydrate and antibiotic metabolism genes were enriched in SA PGPB, which likely facilitated their adaptation to the plant growth environment. Our findings provide genetic insights on LA and SA PGPB strains' ecological adaptation and biocontrol characteristics.