Plant hypersensitive induced reaction protein facilitates cell death induced by secreted xylanase associated with the pathogenicity of Sclerotinia sclerotiorum.

Necrotrophic fungal plant pathogens employ cell death-inducing proteins (CDIPs) to facilitate infection. However, the specific CDIPs and their mechanisms in pathogenic processes of Sclerotinia sclerotiorum, a necrotrophic pathogen that causes disease in many economically important crop species, have not yet been clearly defined. This study found that S. sclerotiorum secretes SsXyl2, a glycosyl hydrolase family 11 xylanase, at the late stage of hyphal infection. SsXyl2 targets the apoplast of host plants to induce cell death independent of xylanase activity. Targeted disruption of SsXyl2 leads to serious impairment of virulence, which can be recovered by a catalytically impaired SsXyl2 variant, thus supporting the critical role of cell death-inducing activity of SsXyl2 in establishing successful colonization of S. sclerotiorum. Remarkably, infection by S. sclerotiorum induces the accumulation of Nicotiana benthamiana hypersensitive-induced reaction protein 2 (NbHIR2). NbHIR2 interacts with SsXyl2 at the plasma membrane and promotes its localization to the cell membrane and cell death-inducing activity. Furthermore, gene-edited mutants of NbHIR2 displayed increased resistance to the wild-type strain of S. sclerotiorum, but not to the SsXyl2-deletion strain. Hence, SsXyl2 acts as a CDIP that manipulates host cell physiology by interacting with hypersensitive induced reaction protein to facilitate colonization by S. sclerotiorum. These findings provide valuable insights into the pathogenic mechanisms of CDIPs in necrotrophic pathogens and lead to a more promising approach for breeding resistant crops against S. sclerotiorum.

XLG2 and CORI3 function additively to regulate plant defense against the necrotrophic pathogen Sclerotinia sclerotiorum.

The membrane-bound heterotrimeric G-proteins in plants play a crucial role in defending against a broad range of pathogens. This study emphasizes the significance of Extra-large Gα protein 2 (XLG2), a plant-specific G-protein, in mediating the plant response to Sclerotinia sclerotiorum, which infects over 600 plant species worldwide. Our analysis of Arabidopsis G-protein mutants showed that loss of XLG2 function increased susceptibility to S. sclerotiorum, accompanied by compromised accumulation of jasmonic acid (JA) during pathogen infection. Overexpression of the XLG2 gene in xlg2 mutant plants resulted in higher resistance and increased JA accumulation during S. sclerotiorum infection. Co-immunoprecipitation (co-IP) analysis on S. sclerotiorum infected Col-0 samples, using two different approaches, identified 201 XLG2-interacting proteins. The identified JA-biosynthetic and JA-responsive proteins had compromised transcript expression in the xlg2 mutant during pathogen infection. XLG2 was found to interact physically with a JA-responsive protein, Coronatine induced 1 (CORI3) in Co-IP, and confirmed using split firefly luciferase complementation and bimolecular fluorescent complementation assays. Additionally, genetic analysis revealed an additive effect of XLG2 and CORI3 on resistance against S. sclerotiorum, JA accumulation, and expression of the defense marker genes. Overall, our study reveals two independent pathways involving XLG2 and CORI3 in contributing resistance against S. sclerotiorum.

A GDSL motif-containing lipase modulates Sclerotinia sclerotiorum resistance in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a devastating disease infecting hundreds of plant species. It also restricts the yield, quality, and safe production of rapeseed (Brassica napus) worldwide. However, the lack of resistance sources and genes to S. sclerotiorum has greatly restricted rapeseed SSR-resistance breeding. In this study, a previously identified GDSL motif-containing lipase gene, Brassica napus GDSL LIPASE-LIKE 1 (BnaC07.GLIP1), encoding a protein localized to the intercellular space, was characterized as functioning in plant immunity to S. sclerotiorum. The BnaC07.GLIP1 promoter is S. sclerotiorum-inducible and the expression of BnaC07.GLIP1 is substantially enhanced after S. sclerotiorum infection. Arabidopsis (Arabidopsis thaliana) heterologously expressing and rapeseed lines overexpressing BnaC07.GLIP1 showed enhanced resistance to S. sclerotiorum, whereas RNAi suppression and CRISPR/Cas9 knockout B. napus lines were hyper-susceptible to S. sclerotiorum. Moreover, BnaC07.GLIP1 affected the lipid composition and induced the production of phospholipid molecules, such as phosphatidylethanolamine, phosphatidylcholine and phosphatidic acid, which were correlated with decreased levels of reactive oxygen species (ROS) and enhanced expression of defense-related genes. A B. napus bZIP44 transcription factor specifically binds the CGTCA motif of the BnaC07.GLIP1 promoter to positively regulate its expression. BnbZIP44 responded to S. sclerotiorum infection, and its heterologous expression inhibited ROS accumulation, thereby enhancing S. sclerotiorum resistance in Arabidopsis. Thus, BnaC07.GLIP1 functions downstream of BnbZIP44 and is involved in S. sclerotiorum resistance by modulating the production of phospholipid molecules and ROS homeostasis in B. napus, providing insights into the potential roles and functional mechanisms of BnaC07.GLIP1 in plant immunity and for improving rapeseed SSR disease-resistance breeding.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

RAS signalling genes can be used as host-induced gene silencing targets to control fungal diseases caused by Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum causes white mold (also called stem rot, Sclerotinia blight, etc.) in many economically important plants. It is a notorious soilborne fungal pathogen due to its wide host range and ability to survive in soil for long periods of time as sclerotia. Although host-induced gene silencing (HIGS) was recently demonstrated to be an effective method for controlling white mold, limited gene targets are available. Here, using a forward genetics approach, we identified a RAS-GTPase activating protein, SsGAP1, which plays essential roles in sclerotia formation, compound appressoria production and virulence. In parallel, as revealed by our knockout analysis, the SsGAP1 ortholog in Botrytis cinerea, BcGAP1, plays similar roles in fungal development and virulence. By knocking down SsRAS1 and SsRAS2, we also revealed that both SsRAS1 and SsRAS2 are required for vegetative growth, sclerotia development, compound appressoria production and virulence in S. sclerotiorum. Due to the major roles these RAS signalling components play in Sclerotiniaceae biology, they can be used as HIGS targets to control diseases caused by both S. sclerotiorum and B. cinerea. Indeed, when we introduced HIGS constructs targeting SsGAP1, SsRAS1 and SsRAS2 in Nicotiana benthamiana and Arabidopsis thaliana, we observed reduced virulence. Taken together, our forward genetics gene discovery pipeline in S. sclerotiorum is highly effective in identifying novel HIGS targets to control S. sclerotiorum and B. cinerea.

Complete genome sequence of Bacillus velezensis strain Ag109, a biocontrol agent against plant-parasitic nematodes and Sclerotinia sclerotiorum.

Soybean is the main oilseed cultivated worldwide. Even though Brazil is the world's largest producer and exporter of soybean, its production is severely limited by biotic factors. Soil borne diseases are the most damaging biotic stressors since they significantly reduce yield and are challenging to manage. In this context, the present study aimed to evaluate the potential of a bacterial strain (Ag109) as a biocontrol agent for different soil pathogens (nematodes and fungi) of soybean. In addition, the genome of Ag109 was wholly sequenced and genes related to secondary metabolite production and plant growth promotion were mined. Ag109 showed nematode control in soybean and controlled 69 and 45% of the populations of Meloidogyne javanica and Pratylenchus brachyurus, respectively. Regarding antifungal activity, these strains showed activity against Macrophomia phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum. For S. sclerotiorum, this strain increased the number of healthy plants and root dry mass compared to the control (with inoculation). Based on the average nucleotide identity and digital DNA-DNA hybridization, this strain was identified as Bacillus velezensis. Diverse clusters of specific genes related to secondary metabolite biosynthesis and root growth promotion were identified, highlighting the potential of this strain to be used as a multifunctional microbial inoculant that acts as a biological control agent while promoting plant growth in soybean.

Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

Selenium in soil enhances resistance of oilseed rape to Sclerotinia sclerotiorum by optimizing the plant microbiome.

Plants can recruit beneficial microbes to enhance their ability to resist disease. It is well established that selenium is beneficial in plant growth, but its role in mediating microbial disease resistance remains poorly understood. Here, we investigated the correlation between selenium, oilseed rape rhizosphere microbes, and Sclerotinia sclerotiorum. Soil application of 0.5 and 1.0 mg kg-1 selenium [selenate Na2SeO4, Se(VI) or selenite Na2SeO3, Se(IV)] significantly increased the resistance of oilseed rape to Sclerotinia sclerotiorum compared with no selenium application, with a disease inhibition rate higher than 20% in Se(VI)0.5, Se(IV)0.5 and Se(IV)1.0 mg kg-1 treatments. The disease resistance of oilseed rape was related to the presence of rhizosphere microorganisms and beneficial bacteria isolated from the rhizosphere inhibited Sclerotinia stem rot. Burkholderia cepacia and the synthetic community consisting of Bacillus altitudinis, Bacillus megaterium, Bacillus cereus, Bacillus subtilis, Bacillus velezensis, Burkholderia cepacia, and Flavobacterium anhui enhanced plant disease resistance through transcriptional regulation and activation of plant-induced systemic resistance. In addition, inoculation of isolated bacteria optimized the bacterial community structure of leaves and enriched beneficial microorganisms such as Bacillus, Pseudomonas, and Sphingomonas. Bacillus isolated from the leaves were sprayed on detached leaves, and it also performed a significant inhibition effect on Sclerotinia sclerotiorum. Overall, our results indicate that selenium improves plant rhizosphere microorganisms and increase resistance to Sclerotinia sclerotiorum in oilseed rape.

Double-Stranded RNA Targeting White Mold Sclerotinia sclerotiorum Argonaute 2 for Disease Control via Spray-Induced Gene Silencing.

Sclerotinia sclerotiorum, the causal agent of white mold infection, is a cosmopolitan fungal pathogen that causes major yield losses in many economically important crops. Spray-induced gene silencing has recently been shown to be a promising alternative method for controlling plant diseases. Based on our prior research, we focused on developing a spray-induced gene silencing approach to control white mold by silencing S. sclerotiorum argonaute 2 (SsAgo2), a crucial part of the fungal small RNA pathway. We compared the lesion size as a result of targeting each ∼500-bp segment of SsAgo2 from the 5' to the 3' end and found that targeting the PIWI/RNaseH domain of SsAgo2 is most effective. External application of double-stranded RNA (dsRNA)-suppressed white mold infection using either in vitro or in vivo transcripts was determined at the rate of 800 ng/0.2 cm2 area with a downregulation of SsAgo2 from infected leaf tissue confirmed by RT-qPCR. Furthermore, magnesium/iron-layered double hydroxide nanosheets loaded with in vitro- and in vivo-transcribed dsRNA segments significantly reduced the rate of S. sclerotiorum lesion expansion. In vivo-produced dsRNA targeting the PIWI/RNaseH domain of the SsAgo2 transcript showed increased efficacy in reducing the white mold symptoms of S. sclerotiorum when combined with layered double hydroxide nanosheets. This approach is promising to produce a large scale of dsRNA that can be deployed as an environmentally friendly fungicide to manage white mold infections in the field.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum.

BACKGROUND: Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. RESULTS: Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. CONCLUSIONS: Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.

Natural α-Methylene-γ-Butyrolactone Analogues: Synthesis and Evaluation of Isoaurone Derivatives as Antifungal Agents.

In order to develop novel, efficient and green fungicides, a series of novel isoaurone derivatives were designed and synthesized, which were characterized by 1H and 13C NMR, high-resolution mass spectra and melting points. The target compounds showed different inhibitory activities against seven plant pathogenic fungi. Compounds 1, 12, 17, 20, 22, 24 and intermediate A showed more than 90% inhibition rates against S. s at 50 mg/L. Interestingly, compound 22 and intermediate A showed the great inhibitory effect against S. s with EC50 values of 4.65 and 4.24 mg/L, which were better than the lead compound isoaurone (EC50 = 15.62 mg/L). The EC50 values of compounds 17 and 24 against B. c were 13.94 and 22.13 mg/L. Moreover, compound 19 displayed significant antifungal activity against G. g with the EC50 value of 11.88 mg/L. Theoretical calculations by DFT revealed that the α, ß-unsaturated carbonyl bond and the benzyl ring are very importantly linked to the strength of the fungicidal activity. Therefore, this study identified a valuable antifungal lead compound for further development of green fungicides.

Exploring the Mycovirus Sclerotinia sclerotiorum Hypovirulence-Associated DNA Virus 1 as a Biocontrol Agent of White Mold Caused by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing white mold on many important economic crops. Recently, some mycoviruses such as S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) converted S. sclerotiorum into a beneficial symbiont that helps plants manage pathogens and other stresses. To explore the potential use of SsHADV-1 as a biocontrol agent in the United States and to test the efficacy of SsHADV-1-infected United States isolates in managing white mold and other crop diseases, SsHADV-1 was transferred from the Chinese strain DT-8 to United States isolates of S. sclerotiorum. SsHADV-1 is readily transmitted horizontally among United States isolates of S. sclerotiorum and consistently conferred hypovirulence to its host strains. Biopriming of dry bean seeds with hypovirulent S. sclerotiorum strains enhanced resistance to white mold, gray mold, and Rhizoctonia root rot. To investigate the underlying mechanisms, endophytic growth of hypovirulent S. sclerotiorum in dry beans was confirmed using PCR, and the expression of 12 plant defense-related genes were monitored before and after infection. The results indicated that the endophytic growth of SsHADV-1-infected strains in plants stimulated the expression of plant immunity pathway genes that assisted a rapid response from the plant to fungal infection. Finally, application of the seed biopriming technology with SsHADV-1-infected hypervirulent strain has promise for the biological control of several diseases of wheat, pea, and sunflower.

Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious and sometimes devastating disease of lupin (Lupinus spp.). A total of 236 lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and "near-immune" responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus, and L. pilosus (totaling ∼4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, 2 accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totaling ∼10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, 1 L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, Walkaway-3 (WW-3; pathotype 10). Against this isolate, five L. angustifolius accessions showed an HR response and four showed an R response, and the L. digitatus accession showed a moderate resistance response. Overall, isolate WW-3 caused significantly (P < 0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a "wild" naturalized field population of L. cosentinii were screened in situ in the field against isolate MBRS-1. Five (∼1.5%) of the 328 plants of wild lupin showed an immune response, 63 (∼19%) showed an HR response, and 146 (∼45%) showed an R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high-level resistance to Sclerotinia stem rot identified in this study can be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, ß-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

A Glycosyl Hydrolase 5 Family Protein Is Essential for Virulence of Necrotrophic Fungi and Can Suppress Plant Immunity.

Phytopathogenic fungi normally secrete large amounts of CWDEs to enhance infection of plants. In this study, we identified and characterized a secreted glycosyl hydrolase 5 family member in Sclerotinia sclerotiorum (SsGH5, Sclerotinia sclerotiorum Glycosyl Hydrolase 5). SsGH5 was significantly upregulated during the early stages of infection. Knocking out SsGH5 did not affect the growth and acid production of S. sclerotiorum but resulted in decreased glucan utilization and significantly reduced virulence. In addition, Arabidopsis thaliana expressing SsGH5 became more susceptible to necrotrophic pathogens and basal immune responses were inhibited in these plants. Remarkably, the lost virulence of the ΔSsGH5 mutants was restored after inoculating onto SsGH5 transgenic Arabidopsis. In summary, these results highlight that S. sclerotiorum suppresses the immune responses of Arabidopsis through secreting SsGH5, and thus exerts full virulence for successful infection.

Glutamate Receptor-like (GLR) Family in Brassica napus: Genome-Wide Identification and Functional Analysis in Resistance to Sclerotinia sclerotiorum.

Plant glutamate receptor-like channels (GLRs) are homologs of animal ionotropic glutamate receptors. GLRs are critical in various plant biological functions, yet their genomic features and functions in disease resistance remain largely unknown in many crop species. Here, we report the results on a thorough genome-wide study of the GLR family in oilseed rape (Brassica napus) and their role in resistance to the fungal pathogen Sclerotinia sclerotiorum. A total of 61 GLRs were identified in oilseed rape. They comprised three groups, as in Arabidopsis thaliana. Detailed computational analyses, including prediction of domain and motifs, cellular localization, cis-acting elements, PTM sites, and amino acid ligands and their binding pockets in BnGLR proteins, unveiled a set of group-specific characteristics of the BnGLR family, which included chromosomal distribution, motif composition, intron number and size, and methylation sites. Functional dissection employing virus-induced gene silencing of BnGLRs in oilseed rape and Arabidopsis mutants of BnGLR homologs demonstrated that BnGLR35/AtGLR2.5 positively, while BnGLR12/AtGLR1.2 and BnGLR53/AtGLR3.2 negatively, regulated plant resistance to S. sclerotiorum, indicating that GLR genes were differentially involved in this resistance. Our findings reveal the complex involvement of GLRs in B. napus resistance to S. sclerotiorum and provide clues for further functional characterization of BnGLRs.

Integrated Assays of Genome-Wide Association Study, Multi-Omics Co-Localization, and Machine Learning Associated Calcium Signaling Genes with Oilseed Rape Resistance to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Ss) is one of the most devastating fungal pathogens, causing huge yield loss in multiple economically important crops including oilseed rape. Plant resistance to Ss pertains to quantitative disease resistance (QDR) controlled by multiple minor genes. Genome-wide identification of genes involved in QDR to Ss is yet to be conducted. In this study, we integrated several assays including genome-wide association study (GWAS), multi-omics co-localization, and machine learning prediction to identify, on a genome-wide scale, genes involved in the oilseed rape QDR to Ss. Employing GWAS and multi-omics co-localization, we identified seven resistance-associated loci (RALs) associated with oilseed rape resistance to Ss. Furthermore, we developed a machine learning algorithm and named it Integrative Multi-Omics Analysis and Machine Learning for Target Gene Prediction (iMAP), which integrates multi-omics data to rapidly predict disease resistance-related genes within a broad chromosomal region. Through iMAP based on the identified RALs, we revealed multiple calcium signaling genes related to the QDR to Ss. Population-level analysis of selective sweeps and haplotypes of variants confirmed the positive selection of the predicted calcium signaling genes during evolution. Overall, this study has developed an algorithm that integrates multi-omics data and machine learning methods, providing a powerful tool for predicting target genes associated with specific traits. Furthermore, it makes a basis for further understanding the role and mechanisms of calcium signaling genes in the QDR to Ss.

Mechanisms of Primed Defense: Plant Immunity Induced by Endophytic Colonization of a Mycovirus-Induced Hypovirulent Fungal Pathogen.

How mycovirus-induced hypovirulence in fungi activates plant defense is still poorly understood. The changes in plant fitness and gene expression caused by the inoculation of the fungus Sclerotinia sclerotiorum harboring and made hypovirulent by the mycovirus soybean leaf-associated gemygorvirus-1 (SlaGemV-1) of the species Gemycircularvirus soybe1 were examined in this study. As the hypovirulent fungus (DK3V) colonized soybean Glycine max, plant transcriptomic analysis indicated changes in defense responses and photosynthetic activity, supported by an upregulation of individual genes and overrepresentation of photosystem gene ontology groups. The upregulated genes include genes relating to both pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity as well as various genes relating to the induction of systemic acquired resistance and the biosynthesis of jasmonic acid. Plants colonized with DK3V showed a resistant phenotype to virulent S. sclerotiorum infection. Plant height and leaf area were also determined to be larger in plants grown with the virus-infected fungus. Here, we hypothesize that inoculation of soybean with DK3V can result in the triggering of a wide range of defense mechanisms to prime against later infection. The knowledge gained from this study about plant transcriptomics and phenotype will help prime plant immunity with mycovirus-infected hypovirulent fungal strains more effectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Role of a Soybean 14-3-3 Gene (Glyma05g29080) on White Mold Resistance and Nodulation Investigations Using CRISPR-Cas9 Editing and RNA Silencing.

The role of a soybean 14-3-3 gene (Glyma05g29080) in defense against white mold and in nodulation was investigated by loss-of-gene-function with CRISPR-Cas9 editing and silencing of RNA interference (RNAi). Particle bombardment was used to introduce the CRISPR expression cassette to target the soybean 14-3-3 gene and an RNAi construct to silence gene transcription. Transmission of the edited 14-3-3 gene and the RNAi construct was confirmed in their respective progeny. The recovered transgenic plants and their progeny were significantly more susceptible to Sclerotinia sclerotiorum infection and showed a significant reduction in nodulation, thus confirming the role of the 14-3-3 gene (Glyma05g29080) in both nodulation and defense.

Screening of microRNAs and target genes involved in Sclerotinia sclerotiorum (Lib.) infection in Brassica napus L.

BACKGROUND: Rapeseed (Brassica napus L.) is the third largest source of vegetable oil in the world, and Sclerotinia sclerotiorum (Lib.) is a major soil-borne fungal plant pathogen that infects more than 400 plant species, including B. napus. Sclerotinia stem rot caused an annual loss of 10 - 20% in rapeseed yield. Exploring the molecular mechanisms in response to S. sclerotiorum infection in B. napus is beneficial for breeding and cultivation of resistant varieties. To gain a better understanding of the mechanisms regarding B. napus tolerance to Sclerotinia stem rot, we employed a miRNAome sequencing approach and comprehensively investigated global miRNA expression profile among five relatively resistant lines and five susceptible lines of oilseed at 0, 24, and 48 h post-inoculation. RESULTS: In this study, a total of 40 known and 1105 novel miRNAs were differentially expressed after S. sclerotiorum infection, including miR156, miR6028, miR394, miR390, miR395, miR166, miR171, miR167, miR164, and miR172. Furthermore, 8,523 genes were predicted as targets for these differentially expressed miRNAs. These target genes were mainly associated with disease resistance (R) genes, signal transduction, transcription factors, and hormones. Constitutively expressing miR156b (OX156b) plants strengthened Arabidopsis resistance against S. sclerotiorum accompanied by smaller necrotic lesions, whereas blocking miR156 expression in Arabidopsis (MIM156) led to greater susceptibility to S. sclerotiorum disease, associated with extensive cell death of necrotic lesions. CONCLUSIONS: This study reveals the distinct difference in miRNA profiling between the relatively resistant lines and susceptible lines of B. napus in response to S. sclerotiorum. The identified differentially expressed miRNAs related to sclerotinia stem rot resistance are involved in regulating resistance to S. sclerotiorum in rapeseed by targeting genes related to R genes, signal transduction, transcription factors, and hormones. miR156 positively modulates the resistance to S. sclerotiorum infection by restricting colonization of S. sclerotiorum mycelia. This study provides a broad view of miRNA expression changes after S. sclerotiorum infection in oilseed and is the first to elucidate the function and mechanism underlying the miR156 response to S. sclerotiorum infection in oilseed rape.