mRNA Turnover Protein 4 Is Vital for Fungal Pathogenicity and Response to Oxidative Stress in Sclerotinia sclerotiorum.

Ribosome assembly factors have been extensively studied in yeast, and their abnormalities may affect the assembly process of ribosomes and cause severe damage to cells. However, it is not clear whether mRNA turnover protein 4 (MRT4) functions in the fungal growth and pathogenicity in Sclerotinia sclerotiorum. Here, we identified the nucleus-located gene SsMRT4 using reverse genetics, and found that knockdown of SsMRT4 resulted in retard mycelia growth and complete loss of pathogenicity. Furthermore, mrt4 knockdown mutants showed almost no appressorium formation and oxalic acid production comparing to the wild-type and complementary strains. In addition, the abilities to ROS elimination and resistance to oxidative and osmotic stresses were also seriously compromised in mrt4 mutants. Overall, our study clarified the role of SsMRT4 in S. sclerotiorum, providing new insights into ribosome assembly in regulating pathogenicity and resistance to environmental stresses of fungi.

An Amidase Contributes to Full Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is one of the most notorious and ubiquitous soilborne plant pathogens, causing serious economic losses to a large number of hosts worldwide. Although virulence factors have been identified in this filamentous fungus, including various cell-wall-degrading enzymes, toxins, oxalic acids and effectors, our understanding of its virulence strategies is far from complete. To explore novel factors contributing to disease, a new pipeline combining forward genetic screening and next-generation sequencing was utilized in this study. Analysis of a hypovirulent mutant revealed that a mutation in an amidase-encoding gene, Sscle_10g079050, resulted in reduced virulence. This is a first report on the contribution of an amidase to fungal virulence, likely through affecting oxalic acid homeostasis.

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.

A Single Amino Acid Substitution in RFC4 Leads to Endoduplication and Compromised Resistance to DNA Damage in Arabidopsis thaliana.

Replication factor C (RFC) is a heteropentameric ATPase associated with the diverse cellular activities (AAA+ATPase) protein complex, which is composed of one large subunit, known as RFC1, and four small subunits, RFC2/3/4/5. Among them, RFC1 and RFC3 were previously reported to mediate genomic stability and resistance to pathogens in Arabidopsis. Here, we generated a viable rfc4e (rfc4-1/RFC4G54E) mutant with a single amino acid substitution by site-directed mutagenesis. Three of six positive T2 mutants with the same amino acid substitution, but different insertion loci, were sequenced to identify homozygotes, and the three homozygote mutants showed dwarfism, early flowering, and a partially sterile phenotype. RNA sequencing revealed that genes related to DNA repair and replication were highly upregulated. Moreover, the frequency of DNA lesions was found to be increased in rfc4e mutants. Consistent with this, the rfc4e mutants were very sensitive to DSB-inducing genotoxic agents. In addition, the G54E amino acid substitution in AtRFC4 delayed cell cycle progression and led to endoduplication. Overall, our study provides evidence supporting the notion that RFC4 plays an important role in resistance to genotoxicity and cell proliferation by regulating DNA damage repair in Arabidopsis thaliana.

SsNEP2 Contributes to the Virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a notorious soilborne fungal pathogen that causes serious economic losses globally. The necrosis and ethylene-inducible peptide 1 (NEP1)-like proteins (NLPs) were previously shown to play an important role in pathogenicity in fungal and oomycete pathogens. Here, we generated S. sclerotiorum necrosis and ethylene-inducible peptide 2 (SsNEP2) deletion mutant through homologous recombination and found that SsNEP2 contributes to the virulence of S. sclerotiorum without affecting the development of mycelia, the formation of appressoria, or the secretion of oxalic acid. Although knocking out SsNEP2 did not affect fungal sensitivity to oxidative stress, it did lead to decreased accumulation of reactive oxygen species (ROS) in S. sclerotiorum. Furthermore, Ssnlp24SsNEP2 peptide derived from SsNEP2 triggered host mitogen-activated protein kinase (MAPK) activation, increased defense marker gene expression, and enhanced resistance to Hyaloperonospora arabidopsidis Noco2. Taken together, our data suggest that SsNEP2 is involved in fungal virulence by affecting ROS levels in S. sclerotiorum. It can serve as a pathogen-associated molecular pattern (PAMP) and trigger host pattern triggered immunity to promote the necrotrophic lifestyle of S. sclerotiorum.

CRMP4 CpG Hypermethylation Predicts Upgrading to Gleason Score ≥ 8 in Prostate Cancer.

Background: This study determined the predictive value of CRMP4 promoter methylation in prostate tissues collected by core needle biopsies for a postoperative upgrade of Gleason Score (GS) to ≥8 in patients with low-risk PCa. Method: A retrospective analysis of the clinical data was conducted from 631 patients diagnosed with low-risk PCa by core needle biopsy at multiple centers and then underwent Radical Prostatectomy (RP) from 2014-2019. Specimens were collected by core needle biopsy to detect CRMP4 promoter methylation. The pathologic factors correlated with the postoperative GS upgrade to ≥8 were analyzed by logistic regression. The cut-off value for CRMP4 promoter methylation in the prostate tissues collected by core needle biopsy was estimated from the ROC curve in patients with a postoperative GS upgrade to ≥8. Result: Multivariate logistic regression showed that prostate volume, number of positive cores, and CRMP4 promoter methylation were predictive factors for a GS upgrade to ≥8 (OR: 0.94, 95% CI: 0.91-0.98, P=0.003; OR: 3.16, 95% CI: 1.81-5.53, P<0.001; and OR: 1.43, 95% CI: 1.32-1.55, P<0.001, respectively). The positive predictive rate was 85.2%, the negative predictive rate was 99.3%, and the overall predictive rate was 97.9%. When the CRMP4 promoter methylation rate was >18.00%, the low-risk PCa patients were more likely to escalate to high-risk patients. The predictive sensitivity and specificity were 86.9% and 98.8%, respectively. The area under the ROC curve (AUC) was 0.929 (95% CI: 0.883-0.976; P<0.001). The biochemical recurrence (BCR)-free survival, progression-free survival (PFS), and cancer-specific survival (CSS) were worse in patients with CRMP4 methylation >18.0% and postoperative GS upgrade to ≥8 than in patients without an upgrade (P ≤ 0.002). Conclusion: A CRMP4 promoter methylation rate >18.00% in prostate cancer tissues indicated that patients were more likely to escalate from low-to-high risk after undergoing an RP. We recommend determining CRMP4 promoter methylation before RP for low-risk PCa patients.