The Transcription Factor CcRlm1 Regulates Cell Wall Maintenance and Poplar Defense Response by Directly Targeting CcChs6 and CcGna1 in Cytospora chrysosperma.

Maintenance of cell wall integrity is important for fungal cell morphology against external stresses and even virulence. Although the transcription factor Rlm1 is known to play major regulatory roles in the maintenance of cell integrity, the underlying mechanism of how Rlm1 contributes to cell wall integrity and virulence in phytopathogenic fungi remains unclear. Here, we demonstrated that CcRlm1 plays important roles in cell wall maintenance and virulence in the poplar canker fungus Cytospora chrysosperma. Among putative downstream targets, CcChs6 (chitin synthase) and CcGna1 (glucosamine 6-phosphate N-acetyltransferase) were found to be direct targets of CcRlm1 and shown to function in chitin synthesis and virulence. Furthermore, we found stronger induction of poplar defense responses when challenged with these gene deletion mutants. Collectively, these results suggest that CcRlm1 plays a critical role in the regulation of cell wall maintenance, stress response, and virulence by directly regulating CcChs6 and CcGna1 in C. chrysosperma. IMPORTANCE Cytospora chrysosperma causes canker diseases on woody plants, and the molecular basis of its infection is not well understood. This study shows that CcRlm1 is the major regulator of chitin synthesis and virulence of the poplar canker fungus. Our research contributes to further understanding the molecular basis of the interaction between C. chrysosperma and poplar.

Participation of a MADS-box transcription factor, Mb1, in regulation of the biocontrol potential in an insect fungal pathogen.

The cell wall is crucial for fungal growth, proliferation and interaction with the environment and host. Understanding the regulatory mechanism of cell wall integrity may help with improvement of fungal biocontrol agents. Here, a putative target of the cell wall integrity pathway-involved Slt2 MAP kinase, Mb1, an orthologue of MADS-box transcription factor Rlm1, was characterized in an economically important insect fungal pathogen, Beauveria bassiana. Mb1 disruption mutant (ΔMb1) displayed reduced growth and increased conidial production on minimal medium but not on rich-nutrient media, which is different from ΔSlt2 to a great extent. Loss of Mb1 resulted in a significant increase in sensitivity to cell wall-perturbing agents (Congo red and calcofluor white), with alteration in cell composition that was inconsistent with ΔSlt2 strain, including increased chitin content and reduced chitin-binding ß-1, 3/1,6-glucan levels in the absence of any stress. Transcription levels of 15 chitin synthesis and metabolism-associated and 17 Pkc1-Slt2 CWI (cell wall integrity) pathway, glucan synthesis, and cell wall remodeling enzyme synthesis-involved genes were significantly increased and repressed in ΔMb1 strain, respectively, some of which were verified to be the targets of Mb1. Insect bioassays revealed decreased virulence for the ΔMb1 strain in both topical and intrahemocoel injection assays. Our results demonstrated that Mb1 control fungal biocontrol potential-associated traits, including growth, conidiation and cell wall integrity, in B. bassiana. The difference of Mb1 and Slt2 in contribution to cell wall integrity is discussed.

Phenotypic plasticity within yeast colonies: differential partitioning of cell fates.

Across many phyla, a common aspect of multicellularity is the organization of different cell types into spatial patterns. In the budding yeast Saccharomyces cerevisiae, after diploid colonies have completed growth, they differentiate to form alternating layers of sporulating cells and feeder cells. In the current study, we found that as yeast colonies developed, the feeder cell layer was initially separated from the sporulating cell layer. Furthermore, the spatial pattern of sporulation in colonies depended on the colony's nutrient environment; in two environments in which overall colony sporulation efficiency was very similar, the pattern of feeder and sporulating cells within the colony was very different. As noted previously, under moderately suboptimal conditions for sporulation-low acetate concentration or high temperature-the number of feeder cells increases as does the dependence of sporulation on the feeder-cell transcription factor, Rlm1. Here we report that even under a condition that is completely blocked sporulation, the number of feeder cells still increased. These results suggest broader implications to our recently proposed "Differential Partitioning provides Environmental Buffering" or DPEB hypothesis.

RLM1, Encoding an R2R3 MYB Transcription Factor, Regulates the Development of Secondary Cell Wall in Rice.

Leaf morphology is an important component of rice ideal plant type. To date, many regulatory genes influencing leaf morphology in rice have been cloned, and their underlying molecular regulatory mechanism has been preliminarily clarified. However, the fine regulation relationship of leaf morphogenesis and plant type remains largely elusive. In this study, a rolling-leaf mutant, named rlm1-D, was obtained and controlled by a pair of dominant nuclear genes. Cytological observations revealed that the rlm1 was mainly caused by abnormal deposition of secondary cell walls. Molecular evidence showed ectopic expression of a MYB-type transcription factor LOC_Os05g46610 was responsible for the phenotype of rlm1-D. A series of experiments, including the transcription factor-centered technology, DNA-binding assay, and electrophoretic mobility shift assay, verified that RLM1 can bind to the promoter of OsCAD2, a key gene responsible for lignin biosynthesis in rice. An interacting partner of RLM1, OsMAPK10, was identified. Multiple biochemical assays confirmed that OsMAPK10 interacted with RLM1. OsMAPK10 positively regulated the lignin content in the leaves and stems of rice. Moreover, OsMAPK10 contributes to RLM1 activation of downstream target genes. In particular, RLM1 is exclusively expressed in the stems at the mature plant stage. The yield of RLM1 knockdown lines increased by over 11% without other adverse agricultural trait penalties, indicating great practical application value. A MAPK-MYB-OsCAD2 genetic regulatory network controlling SCW was proposed, providing a theoretical significance and practical value for shaping the ideal plant type and improving rice yield.

Detection of Blackleg Resistance Gene Rlm1 in Double-Low Rapeseed Accessions from Sichuan Province, by Kompetitive Allele-Specific PCR.

Blackleg is a serious disease in Brassica plants, causing moderate to severe yield losses in rapeseed worldwide. Although China has not suffered from this disease yet (more aggressive Leptosphaeria maculans is not present yet), it is crucial to take provisions in breeding for disease resistance to have excellent blackleg-resistant cultivars already in the fields or in the breeding pipeline. The most efficient strategy for controlling this disease is breeding plants with identified resistance genes. We selected 135 rapeseed accessions in Sichuan, including 30 parental materials and 105 hybrids, and we determined their glucosinolate and erucic acid content and confirmed 17 double-low materials. A recently developed single-nucleotide polymorphism (SNP) marker, SNP_208, was used to genotype allelic Rlm1/rlm1 on chromosome A07, and 87 AvrLm1-resistant materials. Combined with the above-mentioned seed quality data, we identified 11 AvrLm1-resistant double-low rapeseed accessions, including nine parental materials and two hybrids. This study lays the foundation of specific R gene-oriented breeding, in the case that the aggressive Leptosphaeria maculans invades and establishes in China in the future and a robust and less labor consuming method to identify resistance in canola germplasm.

Comparative transcriptome and proteome provide new insights into the regulatory mechanisms of the postharvest deterioration of Pleurotus tuoliensis fruitbodies during storage.

The Pleurotus tuoliensis (Pt), a precious edible mushroom with high economic value, is widely popular for its rich nutrition and meaty texture. However, rapid postharvest deterioration depreciates the commercial value of Pt and severely restricts its marketing. By RNA-Seq transcriptomic and TMT-MS MS proteomic, we study the regulatory mechanisms of the postharvest storage of Pt fruitbodies at 25 ℃ for 0, 38, and 76 h (these three-time points recorded as groups A, B, and C, respectively). 2,008 DEGs (Differentially expressed genes) were identified, and all DEGs shared 265 factors with all DEPs (Differentially expressed proteins). Jointly, the DEGs and DEPs of two-omics showed that the category of the metabolic process contained the most DEGs and DEPs in the biological process by GO (Gene Ontology) classification. The top 17 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways with the highest sum of DEG and DEP numbers in groups B/A (38 h vs. 0 h) and C/A (76 h vs. 0 h) and pathways closely related to energy metabolism were selected for analysis and discussion. Actively expression of CAZymes (Carbohydrate active enzymes), represented by laccase, chitinase, and ß-glucanase, directly leads to the softening of fruitbodies. The transcription factor Rlm1 of 1,3-ß-glucan synthase attracted attention with a significant down-regulation of gene levels in the C/A group. Laccase also contributes, together with phenylalanine ammonia-lyase (PAL), to the discoloration reaction in the first 76 h of the fruitbodies. Significant expression of several crucial enzymes for EMP (Glycolysis), Fatty acid degradation, and Valine, leucine and isoleucine degradation at the gene or protein level supply substantial amounts of acetyl-CoA to the TCA cycle. Citrate synthase (CS), isocitrate dehydrogenase (ICDH), and three mitochondrial respiratory complexes intensify respiration and produce high levels of ROS (Reactive oxygen species) by significant up-regulation. In the ROS scavenging system, only Mn-SOD was significantly up-regulated at the gene level and was probably interacted with Hsp60 (Heat shock protein 60), which was significantly up-regulated at the protein level, to play a dominant role in antioxidation. Three types of stresses - cell wall stress, starvation, and oxidative stress - were suffered by Pt fruitbodies postharvest, resulting in cell cycle arrest and gene expression disorder.

FDA Approved Drug Library Screening Identifies Robenidine as a Repositionable Antifungal.

Due to the increasing prevalence of pathogenic fungal infections, the emergence of antifungal resistant clinical isolates worldwide, and the limited arsenal of available antifungals, developing new antifungal strategies is imperative. In this study, we screened a library of 1068 FDA-approved drugs to identify hits that exhibit broad-spectrum antifungal activity. Robenidine, an anticoccidial agent which has been widely used to treat coccidian infections of poultry and rabbits, was identified in this screen. Physiological concentration of robenidine (8 µM) was able to significantly inhibit yeast cell growth, filamentation and biofilm formation of Candida albicans - the most extensively studied human fungal pathogen. Moreover, we observed a broad-spectrum antifungal activity of this compound against fluconazole resistant clinical isolates of C. albicans, as well as a wide range of other clinically relevant fungal pathogens. Intriguingly, robenidine-treated C. albicans cells were hypersensitive to diverse cell wall stressors, and analysis of the cell wall structure by transmission electron microscopy (TEM) showed that the cell wall was severely damaged by robenidine, implying that this compound may target the cell wall integrity signaling pathway. Indeed, upon robenidine treatment, we found a dose dependent increase in the phosphorylation of the cell wall integrity marker Mkc1, which was decreased after prolonged exposure. Finally, we provide evidence by RNA-seq and qPCR that Rlm1, the downstream transcription factor of Mkc1, may represent a potential target of robenidine. Therefore, our data suggest that robenidine, a FDA approved anti-coccidiosis drug, displays a promising and broadly effective antifungal strategy, and represents a potentially repositionable candidate for the treatment of fungal infections.

[Regulations of RLM1 gene affect the anti-autolytic ability of lager yeast].

The autolysis of brewer's yeast seriously affects the quality of beer and the quality of yeast is considered as one of the key factors in beer brewing. Previous studies on brewer's yeast autolysis showed that RLM1 gene, an important transcription factor in cell integrity pathway, is closely related to the autolysis of yeast. In this study, RLM1 was knocked out and overexpressed in a haploid brewer's yeast. RLM1 disruption resulted in poor anti-autolysis performance of yeast, whereas overexpression of RLM1 contributed to the anti-autolytic ability of yeast. In addition, RLM1 gene knockout affected the osmotic stress resistance, cell wall damage resistance, nitrogen starvation resistance and temperature tolerance of yeast strain. The transcriptional level of GAS1 involved in cell wall assembly and DNA damage response was regulated along with the expression of RLM1, whereas other genes in CWI pathway did not show apparent regularity. RLM1 might mainly affect the expression of GAS1 so as to improve the stress resistance of lager yeast in harsh environment. The result from this study help further understand the mechanism of yeast autolysis and lay a foundation for breeding brewer's yeast strain with better anti-autolytic ability.

How Boundaries Form: Linked Nonautonomous Feedback Loops Regulate Pattern Formation in Yeast Colonies.

Under conditions in which budding yeast form colonies and then undergo meiosis/sporulation, the resulting colonies are organized such that a sharply defined layer of meiotic cells overlays a layer of unsporulated cells termed "feeder cells." This differentiation pattern requires activation of both the Rlm1/cell-wall integrity pathway and the Rim101/alkaline-response pathway. In the current study, we analyzed the connection between these two signaling pathways in regulating colony development by determining expression patterns and cell-autonomy relationships. We present evidence that two parallel cell-nonautonomous positive-feedback loops are active in colony patterning, an Rlm1-Slt2 loop active in feeder cells and an Rim101-Ime1 loop active in meiotic cells. The Rlm1-Slt2 loop is expressed first and subsequently activates the Rim101-Ime1 loop through a cell-nonautonomous mechanism. Once activated, each feedback loop activates the cell fate specific to its colony region. At the same time, cell-autonomous mechanisms inhibit ectopic fates within these regions. In addition, once the second loop is active, it represses the first loop through a cell-nonautonomous mechanism. Linked cell-nonautonomous positive-feedback loops, by amplifying small differences in microenvironments, may be a general mechanism for pattern formation in yeast and other organisms.

The Role of Candida albicans Transcription Factor RLM1 in Response to Carbon Adaptation.

Candida albicans is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of Candida cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of C. albicans RLM1 transcriptional factor. RLM1 is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of C. albicans RLM1 in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of C. albicans cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of C. albicans cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall ß-glucans. In addition, absence of RLM1 has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that RLM1 mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells.