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.

Sclerotinia sclerotiorum utilizes host-derived copper for ROS detoxification and infection.

Necrotrophic plant pathogen induces host reactive oxygen species (ROS) production, which leads to necrosis in the host, allowing the pathogen to absorb nutrients from the dead tissues. Sclerotinia sclerotiorum is a typical necrotrophic pathogen that causes Sclerotinia stem rot in more than 400 species, resulting in serious economic losses. Here, we found that three S. sclerotiorum genes involved in copper ion import/transport, SsCTR1, SsCCS and SsATX1, were significantly up-regulated during infection of Brassica oleracea. Function analysis revealed that these genes involved in fungal ROS detoxification and virulence. On the host side, four genes putatively involved in copper ion homeostasis, BolCCS, BolCCH, BolMT2A and BolDRT112, were significantly down-regulated in susceptible B. oleracea, but stably expressed in resistant B. oleracea during infection. Their homologs were found to promote resistance to S. sclerotiorum and increase antioxidant activity in Arabidopsis thaliana. Furthermore, copper concentration analysis indicated that copper flow from healthy area into the necrotic area during infection. A model was proposed that S. sclerotiorum utilizes host copper to detoxify ROS in its cells, whereas the resistant hosts may restrict the supply of essential copper nutrients to S. sclerotiorum by maintaining copper ion homeostasis during infection.

Investigating genetic relationship of Brassica juncea with B. nigra via virtual allopolyploidy and hexaploidy strategy.

Brassica juncea is an important economic crop of the world; however, the narrow genetic base of this crop has tremendously decreased its crop productivity. As an ancestral species of B. juncea, B. nigra is of great importance in widening the genetic diversity of B. juncea. In the present study, 42 SSR markers were employed to screen the genetic diversity among 83 B. nigra, 16 B. juncea, and other Brassica accessions. The molecular characteristics of 498 virtual B. juncea lines were deduced based on the bands of B. nigra and B. rapa via a virtual allopolyploid strategy, and then compared with natural B. juncea accessions. It was found that B. nigra had rich genetic diversity and could be classified into four subgroups, of which subgroup B-III and subgroup B-IV exhibited the closest and the most distant genetic relationship with B. juncea, respectively. To verify this, a hexaploidy strategy was applied to generated synthetic B. juncea from 20 B. nigra accessions, resulting in 45 new-type B. juncea genotypes. The genetic analyses detected that synthetic B. juncea derived from B. nigra in subgroup B-III was close to natural B. juncea, while B. juncea synthesized with B. nigra from subgroup B-IV exhibited wide genetic diversity and was most distant with current B. juncea. This study revealed a great potential of B. nigra in widening genetic diversity of B. juncea particularly using B. nigra in subgroup B-IV, and is helpful in better understanding of the genetic relationship between B. nigra and B. juncea. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-020-01197-7.

Host-induced gene silencing reveals the role of Sclerotinia sclerotiorum oxaloacetate acetylhydrolase gene in fungal oxalic acid accumulation and virulence.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating necrotrophic pathogen which causes severe yield losses to oilseed production worldwide. Most of efforts at the genetic mitigation of the disease have not been successful. Present investigation was conducted to functionally characterize the effect of down-regulating Ssoah1 during host infection and explore the possibility of boosting host resistance by silencing this gene. We utilized host-induced gene silencing (HIGS) to silence Ssoah1 gene in the S. sclerotiorum fungus. A HIGS based vector was constructed and transformed into Arabidopsis thaliana. The pathogenicity assays in the transgenic A. thaliana lines revealed three T3 transformants with significantly higher resistance to S. sclerotiorum in comparison to untransformed controls. There was a concomitant reduction in expression of Ssoah1 and accumulation of oxalic acid in the necrotic regions of transgenic lines as compared to the non-transgenic controls. Specific Ssoah1-siRNA was highly expressed in HIGS Ssoah1 transgenic lines, as compared with WT and EV plants. The outcomes of oxalic acid estimation revealed that silencing of Ssoah1 results in decreased OA accumulation. The recovered mycelium plugs from HIGS Ssoah1 transgenic lines showed decreased Ssoah1 expression and pathogenesis. These results provide the possibility of using HIGS of Ssoah1 for engineering resistance against S. sclerotiorum.

SsCox17, a copper chaperone, is required for pathogenic process and oxidative stress tolerance of Sclerotinia sclerotiorum.

Stem rot, caused by Sclerotinia sclerotiorum has emerged as one of the major fungal pathogens of oilseed Brassica across the world. The pathogenic development is exquisitely dependent on reactive oxygen species (ROS) modulation. Cox17 is a crucial factor that shuttles copper ions from the cytosol to the mitochondria for the cytochrome c oxidase (CCO) assembly. Currently, no data is available regarding the impact of Cox17 in fungal pathogenesis. The present research was carried out to functionally characterize the role of Cox17 in S. sclerotiorum pathogenesis. SsCox17 transcripts showed high expression levels during inoculation on rapeseed. Intramitochondrial copper content and CCO activity were decreased in SsCox17 gene-silenced strains. The SsCox17 gene expression was up-regulated in the hyphae under oxidative stress and a deficiency response to oxidative stress was detected in SsCox17 gene-silenced strains. Compared to the S. sclerotiorum wild-type strain, there was a concomitant reduction in the virulence of SsCox17 gene-silenced strains. The SsCox17 overexpression strain was further found to increase copper content, CCO activity, tolerance to oxidative stress and virulence. We also observed a certain correlation of appressoria formation and SsCox17. These results provide evidence that SsCox17 is positively associated with fungal virulence and oxidative detoxification.

Phytochemistry, Pharmacology, and Toxicology of Datura Species-A Review.

Datura, a genus of medicinal herb from the Solanaceae family, is credited with toxic as well as medicinal properties. The different plant parts of Datura sp., mainly D. stramonium L., commonly known as Datura or Jimson Weed, exhibit potent analgesic, antiviral, anti-diarrheal, and anti-inflammatory activities, owing to the wide range of bioactive constituents. With these pharmacological activities, D. stramonium is potentially used to treat numerous human diseases, including ulcers, inflammation, wounds, rheumatism, gout, bruises and swellings, sciatica, fever, toothache, asthma, and bronchitis. The primary phytochemicals investigation on plant extract of Datura showed alkaloids, carbohydrates, cardiac glycosides, tannins, flavonoids, amino acids, and phenolic compounds. It also contains toxic tropane alkaloids, including atropine, scopolamine, and hyoscamine. Although some studies on D. stramonium have reported potential pharmacological effects, information about the toxicity remains almost uncertain. Moreover, the frequent abuse of D. stramonium for recreational purposes has led to toxic syndromes. Therefore, it becomes necessary to be aware of the toxic aspects and the potential risks accompanying its use. The present review aims to summarize the phytochemical composition and pharmacological and toxicological aspects of the plant Datura.

Sclerotinia sclerotiorum Thioredoxin1 (SsTrx1) is required for pathogenicity and oxidative stress tolerance.

Sclerotinia sclerotiorum infects host plant tissues by inducing necrosis to source nutrients needed for its establishment. Tissue necrosis results from an enhanced generation of reactive oxygen species (ROS) at the site of infection and apoptosis. Pathogens have evolved ROS scavenging mechanisms to withstand host-induced oxidative damage. However, the genes associated with ROS scavenging pathways are yet to be fully investigated in S. sclerotiorum. We selected the S. sclerotiorum Thioredoxin1 gene (SsTrx1) for our investigations as its expression is significantly induced during S. sclerotiorum infection. RNA interference-induced silencing of SsTrx1 in S. sclerotiorum affected the hyphal growth rate, mycelial morphology, and sclerotial development under in vitro conditions. These outcomes confirmed the involvement of SsTrx1 in promoting pathogenicity and oxidative stress tolerance of S. sclerotiorum. We next constructed an SsTrx1-based host-induced gene silencing (HIGS) vector and mobilized it into Arabidopsis thaliana (HIGS-A) and Nicotiana benthamiana (HIGS-N). The disease resistance analysis revealed significantly reduced pathogenicity and disease progression in the transformed genotypes as compared to the nontransformed and empty vector controls. The relative gene expression of SsTrx1 increased under oxidative stress. Taken together, our results show that normal expression of SsTrx1 is crucial for pathogenicity and oxidative stress tolerance of S. sclerotiorum.

Detection of First Marker Trait Associations for Resistance Against Sclerotinia sclerotiorum in Brassica juncea-Erucastrum cardaminoides Introgression Lines.

A set of 96 Brassica juncea-Erucastrum cardaminoides introgression lines (ILs) were developed with genomic regions associated with Sclerotinia stem rot (Sclerotinia sclerotiorum) resistance from a wild Brassicaceous species E. cardaminoides. ILs were assessed for their resistance responses to stem inoculation with S. sclerotiorum, over three crop seasons (season I, 2011/2012; II, 2014/2015; III, 2016-2017). Initially, ILs were genotyped with transferable SSR markers and subsequently through genotyping by sequencing. SSR based association mapping identified six marker loci associated to resistance in both A and B genomes. Subsequent genome-wide association analysis (GWAS) of 84 ILs recognized a large number of SNPs associated to resistance, in chromosomes A03, A06, and B03. Chromosomes A03 and A06 harbored the maximum number of resistance related SNPs. Annotation of linked genomic regions highlighted an array of resistance mechanisms in terms of signal transduction pathways, hypersensitive responses and production of anti-fungal proteins and metabolites. Of major importance was the clustering of SNPs, encoding multiple resistance genes on small regions spanning approximately 885 kb region on chromosome A03 and 74 kb on B03. Five SNPs on chromosome A03 (6,390,210-381) were associated with LRR-RLK (receptor like kinases) genes that encode LRR-protein kinase family proteins. Genetic factors associated with pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) were predicted on chromosome A03, exhibiting 11 SNPs (6,274,763-994). These belonged to three R-Genes encoding TIR-NBS-LRR proteins. Marker trait associations (MTAs) identified will facilitate marker assisted introgression of these critical resistances, into new cultivars of B. juncea initially and, subsequently, into other crop Brassica species.

Molecular and genetic analysis of defensive responses of Brassica juncea - B. fruticulosa introgression lines to Sclerotinia infection.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a major disease of crop brassicas, with inadequate variation for resistance in primary gene pools. We utilized a wild Brassicaceae species with excellent resistance against stem rot to develop a set of B. juncea - B. fruticulosa introgression lines (ILs). These were assessed for resistance using a highly reproducible stem inoculation technique against a virulent pathogen isolate. Over 40% of ILs showed higher levels of resistance. IL-43, IL-175, IL-215, IL-223 and IL-277 were most resistant ILs over three crop seasons. Sequence reads (21x) from the three most diverse ILs were then used to create B. juncea pseudomolecules, by replacing SNPs of reference B. juncea with those of re-sequenced ILs. Genotyping by sequencing (GBS) was also carried out for 88 ILs. Resultant sequence tags were then mapped on to the B. juncea pseudomolecules, and SNP genotypes prepared for each IL. Genome wide association studies helped to map resistance responses to stem rot. A total of 13 significant loci were identified on seven B. juncea chromosomes (A01, A03, A04, A05, A08, A09 and B05). Annotation of the genomic region around identified SNPs allowed identification of 20 candidate genes belonging to major disease resistance protein families, including TIR-NBS-LRR class, Chitinase, Malectin/receptor-like protein kinase, defensin-like (DEFL), desulfoglucosinolate sulfotransferase protein and lipoxygenase. A majority of the significant SNPs could be validated using whole genome sequences (21x) from five advanced generation lines being bred for Sclerotinia resistance as compared to three susceptible B. juncea germplasm lines. Our findings not only provide critical new understanding of the defensive pathway of B. fruticulosa resistance, but will also enable development of marker candidates for assisted transfer of introgressed resistant loci in to agronomically superior cultivars of crop Brassica.

Mapping resistance responses to Sclerotinia infestation in introgression lines of Brassica juncea carrying genomic segments from wild Brassicaceae B. fruticulosa.

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014-15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.