Sclerotiorum Resistance Research Articles

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.

Disease resistance of Brassica juncea to Sclerotinia sclerotiorum is established through the induction of indole glucosinolate biosynthesis

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the main disease threat to oilseeds in Brassiceae, causing significant yield losses and reduction in oil content and quality. While the genetic mechanism underlying its lethal phenotype, particularly in B. juncea, remains largely unexplored. Transcriptome analysis revealed a large number of defense-related genes and response processes in B. napus and B. oleracea. However, similarities and differences in the defense responses to S. sclerotiorum on B. juncea are rarely reported. In the present study, we reported a B. juncea breeding line of H83 with high S. sclerotiorum resistance, which was used for transcriptome analysis compared to L36 with low resistance. A novel regulatory network was proposed to defend against S. sclerotiorum invasion in B. juncea. Upon infection of S. sclerotiorum, a series of were initiated within 12 h, and then defenses were activated to restrict the development and spread of S. sclerotiorum by inducing the massive synthesis of indole glucosinolates after 24 h. Twelve hub genes involved in the network were identified by the weighted gene co-expression network (WGCNA), which are involved in plant-pathogen interaction, signaling pathway genes, indole glucosinolate biosynthesis and cell wall formation. The hub genes were further validated by qRT-PCR. The research revealed a new resistant line of H83 against S. sclerotiorum and a different regulatory network in B. juncea, which would be beneficial for the future effective breeding of Sclerotinia-resistant varieties.

Pangenome characterization and analysis of the NAC gene family reveals genes for Sclerotinia sclerotiorum resistance in sunflower (Helianthus annuus).

Sunflower (Helianthus annuus) is one of the most important economic crops in oilseed production worldwide. The different cultivars exhibit variability in their resistance genes. The NAC transcription factor (TF) family plays diverse roles in plant development and stress responses. With the completion of the H. annuus genome sequence, the entire complement of genes coding for NACs has been identified. However, the reference genome of a single individual cannot cover all the genetic information of the species. Considering only a single reference genome to study gene families will miss many meaningful genes. Apangenome-wide surveyand characterization of the NAC genes in sunflower species were conducted. In total, 139 HaNAC genes are identified, of which 114 are core and 25 are variable. Phylogenetic analysis of sunflower NAC proteins categorizes these proteins into 16 subgroups. 138 HaNACs are randomly distributed on 17 chromosomes. SNP-based haplotype analysis shows haplotype diversity of the HaNAC genes in wild accessions is richer than in landraces and modern cultivars. Ten HaNAC genes in the basal stalk rot (BSR) resistance quantitative trait loci (QTL) are found. A total of 26 HaNAC genes are differentially expressed in response to Sclerotinia head rot (SHR). A total of 137 HaNAC genes are annotated in Gene Ontology (GO) and are classified into 24 functional groups. GO functional enrichment analysis reveals that HaNAC genes are involved in various functions of the biological process. We identified NAC genes in H. annuus (HaNAC) on a pangenome-wide scale and analyzed S. sclerotiorum resistance-related NACs. This study provided a theoretical basis for further genomic improvement targeting resistance-related NAC genes in sunflowers.

Genome-wide mapping and genomic prediction conditioning sclerotinia stem rot resistance in different ecotypes of Brassica napus (L.) germplasm collections

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a destructive disease in canola that significantly reduces seed yield and oil quality. This research aimed to identify the genomic regions and evaluate the potential of genomic prediction for S. sclerotiorum resistance by screening a diverse panel of 143 spring (SP), and 152 semi-winter and winter (SwW) populations through two rounds of plant inoculation during the full flowering stage. A comprehensive phenotypic evaluation involving four distinct traits identified several lines as potential sources of resistance, which showed superior resistance compared to known resistant check cultivars. A Genome-wide association (GWA) study using two different algorithms identified 29 and 19 SNPs in the SP and SwW populations, respectively, with two or more traits were regarded as significant. Ten and seven significant SNPs from SP and SwW populations, respectively, were co-localized with the previously reported SNPs/QTLs by linkage/GWA mapping studies. Within the regions flanking the significant SNPs, we identified a total of 33 and 22 putative candidate genes related to plant defense responses in the SP and SwW populations, respectively. The genomic prediction (GP) revealed higher predictive ability for the SP population (0.46–0.57) over the SwW population (0.10–0.19), depending on trait specifications. In summary, our study revealed that favorable alleles from significant SNPs from GWA mapping could be utilized as valuable resources for introducing and pyramiding resistance alleles to enhance SSR resistance in canola. Genomic selection could also be used as a potential genomics-assisted approach to improve SSR resistance in canola.

LESION MIMIC MUTANT 1 confers basal resistance to Sclerotinia sclerotiorum in rapeseed via a salicylic acid-dependent pathway.

Rapeseed (Brassica napus) is a major edible oilseed crop consumed worldwide. However, its yield is seriously affected by infection from the broad-spectrum non-obligate pathogen Sclerotinia sclerotiorum due to a lack of highly resistant germplasm. Here, we identified a Sclerotinia-resistant and light-dependent lesion mimic mutant from an ethyl methanesulfonate-mutagenized population of the rapeseed inbred Zhongshuang 11 (ZS11) named lesion mimic mutant 1 (lmm1). The phenotype of lmm1 is controlled by a single recessive gene, named LESION MIMIC MUTANT 1 (LMM1), which mapped onto chromosome C04 by bulked segregant analysis within a 2.71-Mb interval. Histochemical analysis indicated that H2O2 strongly accumulated and cell death occurred around the lesion mimic spots. Among 877 differentially expressed genes (DEGs) between ZS11 and lmm1 leaves, 188 DEGs were enriched in the defense response, including 95 DEGs involved in systemic acquired resistance, which is consistent with the higher salicylic acid levels in lmm1. Combining bulked segregant analysis and transcriptome analysis, we identified a significantly up-regulated gene, BnaC4.PR2, which encodes β-1,3-glucanase, as the candidate gene for LMM1. Overexpression of BnaC4.PR2 may induce a reactive oxygen species burst to trigger partial cell death and systemic acquired resistance. Our study provides a new genetic resource for S. sclerotiorum resistance as well as new insights into disease resistance breeding in B. napus.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Downregulation of Brassica napus MYB69 (BnMYB69) increases biomass growth and disease susceptibility via remodeling phytohormone, chlorophyll, shikimate and lignin levels.

MYB transcription factors are major actors regulating plant development and adaptability. Brassica napus is a staple oil crop and is hampered by lodging and diseases. Here, four B. napus MYB69 (BnMYB69s) genes were cloned and functionally characterized. They were dominantly expressed in stems during lignification. BnMYB69 RNA interference (BnMYB69i) plants showed considerable changes in morphology, anatomy, metabolism and gene expression. Stem diameter, leaves, roots and total biomass were distinctly larger, but plant height was significantly reduced. Contents of lignin, cellulose and protopectin in stems were significantly reduced, accompanied with decrease in bending resistance and Sclerotinia sclerotiorum resistance. Anatomical detection observed perturbation in vascular and fiber differentiation in stems, but promotion in parenchyma growth, accompanied with changes in cell size and cell number. In shoots, contents of IAA, shikimates and proanthocyanidin were reduced, while contents of ABA, BL and leaf chlorophyll were increased. qRT-PCR revealed changes in multiple pathways of primary and secondary metabolisms. IAA treatment could recover many phenotypes and metabolisms of BnMYB69i plants. However, roots showed trends opposite to shoots in most cases, and BnMYB69i phenotypes were light-sensitive. Conclusively, BnMYB69s might be light-regulated positive regulators of shikimates-related metabolisms, and exert profound influences on various internal and external plant traits.

High Sclerotinia sclerotiorum resistance in rapeseed plant has been achieved by OsPGIP6.

Sclerotinia sclerotiorum, a worldwide distributed fungal pathogen, causes serious adverse effects on the yield and seed quality of rapeseed. Polygalacturonase-inhibiting proteins (PGIPs) can protect the cell wall from degradation by pathogen-secreted polygalacturonases (PGs). The present study found several PGIPs from Oryza sativa, especially OsPGIP6 and 3 have much higher inhibitory activities to SsPGs than BnPGIP2 from Brassica napus. Among them, OsPGIP1, 4, 6 can significantly elevate the resistance of transgenic Arabidopsis to S. sclerotiorum. Subsequently, OsPGIP1, 3, 4, 6 were subjected to SSR resistance assay in transgenic rapeseed plants. Among which, OsPGIP6 showed the highest resistance to S. sclerotiorum. At 48 h after detached leaves inoculation, the lesion area of OE-OsPGIP6 rapeseed plants is only 17.93% of the non-transgenic line, and 22.17, 21.32, 52.78, 56.47%, compared to OE-BnPGIP2, OE-OsPGIP1, OE-OsPGIP2, OE-OsPGIP4, respectively. Furthermore, the lesion area of OE-OsPGIP6 reached 10.11% compared to WT at 72 hpi. Also, the lesion length on the stem of OE-OsPGIP6 plants was reduced by 36.83% compared to WT. These results reveal that OsPGIP family, especially OsPGIP6, has a great potential in rapeseed S. sclerotiorum-resistance breeding.

Identification of receptor-like proteins induced by Sclerotinia sclerotiorum in Brassica napus.

Heightening the resistance of plants to microbial infection is a widely concerned issue, especially for economical crops. Receptor-like proteins (RLPs), typically with tandem leucine-rich repeats (LRRs) domain, play a crucial role in mediating immune activation, being an indispensable constituent in the first layer of defense. Based on an analysis of orthologs among Brassica rapa, Brassica oleracea, and Brassica napus using Arabidopsis thaliana RLPs as a reference framework, we found that compared to A. thaliana, there were some obvious evolutionary diversities of RLPs among the three Brassicaceae species. BnRLP encoding genes were unevenly distributed on chromosomes, mainly on chrA01, chrA04, chrC03, chrC04, and chrC06. The orthologs of five AtRLPs (AtRLP3, AtRLP10, AtRLP17, AtRLP44, and AtRLP51) were highly conserved, but retrenchment and functional centralization occurred in Brassicaceae RLPs during evolution. The RLP proteins were clustered into 13 subgroups. Ten BnRLPs presented expression specificity between R and S when elicited by Sclerotinia sclerotiorum, which might be fabulous candidates for S. sclerotiorum resistance research.

Genome-wide identification and functional analysis of cupin_1 domain-containing members involved in the responses to Sclerotinia sclerotiorum and abiotic stress in Brassica napus

Cupin_1 domain-containing proteins (CDPs) are ubiquitously present in higher plants, which are known to play essential roles in various biological processes. In this study, we carried out genome-wide characterization and systematic investigation of the CDP genes in Brassica napus. A total of 96 BnCDPs, including 71 germin-like proteins (GLPs; proteins with a single cupin_1 domain) and 25 CDP bicupins (proteins with two cupin_1 domains), were identified and clustered into six distinct subfamilies (I–VI) based on the phylogenic analysis, gene structure and motif distribution. Further analysis indicated that whole-genome duplication (WGD) and segmental duplication are main contributors to the species-specific expansion of the BnCDP gene family, and all the duplicated genes subsequently underwent strong purification selection. The promoter region of BnCDPs showed enrichment of cis-regulatory elements associated with development, hormone and stress, as well as transcription factor binding sites, which validates the prediction that BnCDPs are widely involved in plant growth and biotic and abiotic stress responses. The BnCDPs in different subfamilies exhibited obvious differences in expression among 30 developmental tissues/stages of B. napus, implying that BnCDPs may be involved in tissue- and stage-specific developmental processes. Similar trends in expression of most BnCDPs were observed under Sclerotinia sclerotiorum inoculation and four abiotic stresses (dehydration, cold, ABA and salinity), particularly the BnGLPs in subfamily I and III with single cupin_1 domain, revealing that BnCDPs are of great importance in the environmental adaption of B. napus. We then performed a genome-wide association study (GWAS) of 274 B. napus core germplasms on S. sclerotiorum resistance and identified four significantly associated loci harboring five BnGLPs. The expression levels of two candidate genes, BnGLP1.A08 and BnGLP1.C08, were significantly correlated with S. sclerotiorum resistance. Their functional responses to multiple stages of S. sclerotiorum inoculation and four abiotic stresses were further examined through qPCR. Overall, this study provides rich resources for research on the function and evolutionary playground of CDP genes.

Early oxidative burst and anthocyanin-mediated antioxidant defense mechanism impart resistance against Sclerotinia sclerotiorum in Indian mustard

The current study builds on our prior findings that anthocyanin buildup around the pathogen inoculation site confers resistance to S. sclerotiorum resistance in Indian mustard. However, the mechanism by which anthocyanin accumulation confers resistance is largely unknown. To better understand this mechanism, we compared the disease responses and biochemical profiling of two contrasting and wild type genotypes of Indian mustard [Sclerotinia stem rot-resistant RH 1222–28 (WR) and susceptible Varuna (WS)], as well as two anthocyanin contrasting mutants [anthocyanin-more (PM) and anthocyanin-devoid (AM) mutant] during different infection phases. Regardless of the inoculation time, disease examination revealed that WR and PM had considerably (P ≤ 0.05) lower mean lesion length (MLL) and rate of lesion expansion than WS and AM. Compared to WS and AM, biochemical results showed that WR and PM responses to infection were earlier, more timely regulated, and more robust. In the resistant WR and PM, hydrogen peroxide (H2O2) and superoxide anions (O2−1) accumulated rapidly and peaked during the early infection stage (2 days after inoculation; DAI), whereas in the susceptible WS and AM, accumulation was stronger and more intense in the later infection stage (8 DAI). Compared to the susceptible genotype (WS), the resistant genotype (WR) had significantly higher anthocyanin accumulation, a lower rate of lipid peroxidation, and a strong, efficient, and timely regulated antioxidant system that limited pathogen colonization and disease development. The anthocyanin-rich mutant (PM) is more resistant to ROS-induced oxidative damage. It has a slower rate of lesion development than the anthocyanin-deficient (AM) mutant, which has lower anthocyanin accumulation and higher oxidative stress with weak antioxidative potential, resulting in significantly (P ≤ 0.05) longer stem lesion length and rate of lesion expansion. These findings imply that during distinct infection phases of S. sclerotiorum, a well-coordinated oxidants/antioxidants system in terms of intensity and timing regulates Sclerotinia stem rot resistance/susceptibility. Overall, anthocyanin accumulation around the pathogen infection site appears to play a critical role in altering the ROS producing and scavenging machinery in Indian mustard, which contributes to resistance to S. sclerotiorum.

QTL Identification for Stem Fiber, Strength and Rot Resistance in a DH Population from an Alien Introgression of Brassica napus.

Stem fiber, stem strength and stem-rot resistance are important agronomic traits in Brassica napus. To understand the molecular mechanism that controls the stem-related traits, we investigated the stem lignin (ADL), cellulose (Cel), hemicellulose (Hem) content, S/G monolignol ratio (SG), stem breaking force (BF), breaking strength (F) and Sclerotinia sclerotiorum resistance (SSR). Each trait was significantly positively or negatively correlated with more than three of the other six traits. QTL mapping for ADL, Cel, Hem, SG, BF, F and SSR were performed using a doubled haploid population derived from an intertribal B. napus introgression line ‘Y689′ crossed with B. napus cv. ‘Westar’. A total of 67 additive QTL were identified and integrated into 55 consensus QTL by meta-analysis. Among the 55 consensus QTL, 23 (41.8%) QTL were co-located and were integrated into 11 unique QTL. The QTL by environment (Q × E) interactions were analyzed and 22 combined QTL were identified. In addition, candidate genes within the QTL intervals were proposed based on the known function of Arabidopsis orthologs. These results provided valuable information for improving lodging resistance, S. sclerotiorum resistance and mechanized harvesting of B. napus.

Melatonin regulated glucosinolate profile via modulation of genes related with sulfur and nitrogen metabolism in Brassica rapa ssp. pekinensis

Melatonin (MT) is a multi–regulatory molecule eliciting pleiotropic effects on plant growth and development. Glucosinolates (GS) are a group of sulfur (S)-rich and nitrogen (N)-containing secondary metabolites that play an important role in plant defense. Although MT treatment is known to strongly induce expression of GS metabolism related genes, it still remains unclear how MT modulates GS profile. In order to better understand the molecular mechanisms of MT-regulated GS profile in Brassica rapa, comparative transcriptomics and HPLC analyses were conducted. Upon MT treatment, overall GS accumulation increased 3.2-fold, along with emergence of three new GS of glucocochlearin (GCR), glucoerucin (GEC) and 4-hydroxyglucobrassicin (4-OHGBC), and aliphatic GS was the most abundant form constituting 63% of the total GS content. Besides, MT treatment generated eleven differentially expressed genes involved in GS biosynthesis, among which eight encode GS pathway enzymes, and three encode MYB transcription factors modulating expression of GS biosynthesis genes. This was accompanied by significantly elevated expression of three S assimilation-related genes and glutamine synthetase gene involved in N assimilation. In vitro disk-diffusion bioassay demonstrated that lyophilized powder of MT-treated rosette leaf, as well as hydrolyzed products of GCR and GEC significantly inhibited the growth of Sclerotinia sclerotiorum. There was a statistically significant negative correlation between 4-OHGBC and colony growth rate, and between 4-methoxyglucobrassicin (4MeGBC) and sclerotia number. Taken together, the results suggested that exogenous MT treatment altered GS profiles and contents by activating the expression of the primary metabolism genes for S and N assimilation, as well as the secondary metabolism genes for GS biosynthesis, thereby playing a prominent role in S. sclerotiorum resistance in B. rapa. MT as an amphiphilic bio-substance, which can easily pass though the cell membrane, may offer a new integrated strategy for ecofriendly and sustainable management to control plant diseases.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate‐5‐hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate‐5‐hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D‐NMR profiling in KO‐7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Identification of Sources of Sclerotinia sclerotiorum Resistance in a Collection of Wild Cicer Germplasm.

Sclerotinia sclerotiorum is an important fungal pathogen of chickpea (Cicer arietinum L.), and it can cause yield losses up to 100%. The wild progenitors are much more diverse than domesticated chickpea, and this study describes how this relates to S. sclerotiorum resistance. Initially, the pathogenicity of nine Australian S. sclerotiorum isolates was examined on three Cicer lines to develop a robust phenotyping assay, and significant differences in isolate aggressiveness were identified with six isolates being classed as highly aggressive and three as moderately aggressive. We identified two S. sclerotiorum isolates, CU8.20 and CU10.12, to be highly aggressive and moderately aggressive, respectively. A subsequent phenotyping assay was conducted using the two isolates to evaluate 86 wild Cicer accessions (Cicer reticulatum and Cicer echinospermum) and two C. arietinum varieties for resistance to S. sclerotiorum. A subset of 12 genotypes was further evaluated, and subsequently, two wild Cicer accessions with consistently high levels of resistance to S. sclerotiorum were examined using the initially characterized nine isolates. Wild Cicer accessions Karab_084 and Deste_063 demonstrated consistent partial resistance to S. sclerotiorum. There were significant differences in responses to S. sclerotiorum across wild Cicer collection sites. The Cermik, Karabahce, and Destek sites' responses to the aggressive isolate CU8.20 ranged from resistant to susceptible, highlighting an interaction between isolate genotype and chickpea collection site for sclerotinia stem rot resistance. This is the first evidence of partial stem resistance identified in wild Cicer germplasm, which can be adopted in chickpea breeding programs to enhance S. sclerotiorum resistance in future chickpea varieties.

Brassica napus Mediator Subunit16 Induces BnMED25- and BnWRKY33-Activated Defense Signaling to Confer Sclerotinia sclerotiorum Resistance.

The plant mediator is a highly conserved protein complex that interacts with transcription factors (TFs) and RNA polymerase II (RNAP II) to relay regulatory information during transcription. Plant immune response is one of the biological processes that is orchestrated by this regulatory mechanism. Brassica napus, an important oil crop, is severely attacked by a devastating disease Sclerotinia stem rot. Here, we explored broad-spectrum disease resistant roles of B. napus mediator subunit 16 (BnMED16) and its host defense mechanism against fugal pathogen Sclerotinia sclerotiorum. We found that BnMED16 expression was significantly increased by S. sclerotiorum infection, and its homologous overexpression resulted in rapid and comprehensive defense responses from the beginning to the end. This affected signal transduction with multiple channels including pathogen recognition, intracellular Ca2+ concentration, reactive oxygen species (ROS) accumulation and clearance, and activation of mitogen-activated protein kinase (MAPK) signaling cascades initially. Subsequently, pathogen-/defense-related genes and hormone-responsive pathways were highly activated, which resulted in enhanced cell wall and secretion of defense proteases. Furthermore, the biochemical analysis showed that BnMED16 interacts with BnMED25 and BnWRKY33. Additionally, BnMED25 also interacts with TFs BnMYC2, BnCOI1, and BnEIN3 of the JA/ET signal transduction pathway. Taken together, we proposed a hypothetical model that BnMED16 confers S. sclerotiorum resistance by enhancing BnMED25-mediated JA/ET defense pathways and BnWRKY33-activated defense signaling in B. napus. The BnMED16 overexpressing lines with enhanced broad-spectrum disease resistance could be useful for breeding Sclerotinia-resistant oilseed rape varieties, as well as serving as basis for further strategy development in resistance breeding.

Melatonin elevated Sclerotinia sclerotiorum resistance via modulation of ATP and glucosinolate biosynthesis in Brassica rapa ssp. pekinensis

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SignificanceMelatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

Modeling first order additive × additive epistasis improves accuracy of genomic prediction for sclerotinia stem rot resistance in canola.

The fungus Sclerotinia sclerotiorum infects hundreds of plant species including many crops. Resistance to this pathogen in canola (Brassica napus L. subsp. napus) is controlled by numerous quantitative trait loci (QTL). For such polygenic traits, genomic prediction may be useful for breeding as it can capture many QTL at once while also considering nonadditive genetic effects. Here, we test application of common regression models to genomic prediction of S. sclerotiorum resistance in canola in a diverse panel of 218 plants genotyped at 24,634 loci. Disease resistance was scored by infection with an aggressive isolate and monitoring over 3 wk. We found that including first-order additive × additive epistasis in linear mixed models (LMMs) improved accuracy of breeding value estimation between 3 and 40%, depending on method of assessment, and correlation between phenotypes and predicted total genetic values by 14%. Bayesian models performed similarly to or worse than genomic relationship matrix-based models for estimating breeding values or overall phenotypes from genetic values. Bayesian ridge regression, which is most similar to the genomic relationship matrix-based approach in the amount of shrinkage it applies to marker effects, was the most accurate of this family of models. This confirms several studies indicating the highly polygenic nature of sclerotinia stem rot resistance. Overall, our results highlight the use of simple epistasis terms for prediction of breeding values and total genetic values for a complex disease resistance phenotype in canola.

Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase: implications for rapeseed resistance to fungal infection.

BnPGIPs interacted with Sclerotinia sclerotiorum PGs to improve rapeseed SSR resistance at different levels; the BnPGIP-overexpression lines did not affect plant morphology or seed quality traits. Plant polygalacturonase-inhibiting proteins (PGIPs) play a crucial role in plant defence against phytopathogenic fungi by inhibiting fungal polygalacturonase (PG) activity. We overexpressed BnPGIP2, BnPGIP5, and BnPGIP10 genes in an inbred line 7492 of rapeseed (Brassica napus). Compared with 7492WT, the overexpression of BnPGIP2 lines significantly increased Sclerotinia sclerotiorum resistance in both seedlings and adult plants. BnPGIP5 overexpression lines exhibited decreased S. sclerotiorum disease symptoms in seedlings only, whereas BnPGIP10 overexpression lines did not improve Sclerotinia resistance for seedlings or adult plants. Quantitative real-time PCR analysis of S. sclerotiorum PG1, SsPG3, SsPG5, and SsPG6 genes in overexpressing BnPGIP lines showed that these pathogenic genes in the Sclerotinia resistance transgenic lines exhibited low expression in stem tissues. Split-luciferase complementation experiments confirmed the following: BnPGIP2 interacts with SsPG1 and SsPG6 but not with SsPG3 or SsPG5; BnPGIP5 interacts with SsPG3 and SsPG6 but not with SsPG1 or SsPG5; and BnPGIP10 interacts with SsPG1 but not SsPG3, SsPG5, or SsPG6. Leaf crude protein extracts from BnPGIP2 and BnPGIP5 transgenic lines displayed high inhibitory activity against the SsPG crude protein. BnPGIP-overexpression lines with Sclerotinia resistance displayed a lower accumulation of H2O2 and higher expression of the H2O2-removing gene BnAPX (ascorbate peroxidase) than 7492WT, as well as elevated expression of defence response genes including jasmonic acid/ethylene and salicylic acid pathways after S. sclerotiorum infection. The plants overexpressing BnPGIP exhibited no difference in either agronomic traits or grain yield from 7492WT. This study provides potential target genes for developing S. sclerotiorum resistance in rapeseed.

The complexity of the Sclerotinia sclerotiorum pathosystem in soybean: virulence factors, resistance mechanisms, and their exploitation to control Sclerotinia stem rot

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a globally important, yield limiting disease of soybean. Progress has been made in our understanding of this pathosystem at the plant level, such as the key role of oxalic acid in disease development and the importance of cell wall-degrading enzymes and other secreted proteins. Unfortunately, advances have largely focused on the fungal side of this interaction and only provide glimpses into the plant mechanisms governing resistance to this pathogen. With the absence of commercially available resistant soybeans, chemical and cultural solutions are being used by farmers to manage SSR with limited success. Additional research is needed to identify S. sclerotiorum resistance mechanisms that can be exploited to improve genetic resistance in soybean and decrease reliance on spray regimes. Technologies such as transgenics and RNAi could be exploited to improve the level of resistance to S. sclerotiorum in soybean. This review offers insight into the hurdles of managing SSR at the plant level and potential solutions that might be adopted in the future.