New N-acylated aminoalkanoic acids from tea roots derived biocontrol agent Clonostachys rosea 15020.

Four new N-acylated aminoalkanoic acids, namely clonoroseins E-H (1-4), together with three previously identified analogs, clonoroseins A, B, and D (5-7), were identified from the endophytic fungus Clonostachys rosea strain 15020 (CR15020), using Feature-based Molecular Networking (FBMN). The elucidation of their chemical structures, including their absolute configurations, was achieved through spectroscopic analysis combined with quantum chemical calculations. Bioinformatics analyses suggested that an iterative type I HR-PKS (CrsE) generates the polyketide side chain of these clonoroseins. Furthermore, a downstream adenylate-forming enzyme of the PKS (CrsD) was suspected to function as an amide synthetase. CrsD potentially facilitates the transformation of the polyketide moiety into an acyl-AMP intermediate, followed by nucleophilic substitution with either ß-alanine or γ-aminobutyric acid to produce amide derivatives. These findings significantly expand our understanding of PKS-related products originating from C. rosea and also underscore the powerful application of FBMN analytical methods in characterization of new compounds.

Diversity of Colletotrichum species associated with anthracnose on Euonymus japonicus and their sensitivity to fungicides.

As an evergreen shrub, Euonymus japonicus plays a crucial role in urban landscape construction, and its growth is affected by severe foliar anthracnose caused by Colletotrichum spp. However, the biodiversity of Colletotrichum species associated with anthracnose on E. japonicus remains undetermined. This study involved a two-year collection of E. japonicus leaf samples with typical anthracnose symptoms from 9 districts in Beijing, China. A total of 194 Colletotrichum isolates were obtained, and eight Colletotrichum species were subsequently identified using morphological characteristics and molecular identification with the ACT, GADPH, CHS, TUB2, and CAL genes, as well as the rDNA-ITS region. These species included Colletotrichum aenigma, C. fructicola, C. gloeosporioides, C. grossum, C. hebeiense, C. karstii, C. siamense, and C. theobromicola with C. siamense being the most prevalent (57%), followed by C. aenigma and C. theobromicola. Furthermore, C. fructicola, C. grossum and C. hebeiense are reported for the first time as causal agents of anthracnose on E. japonicus worldwide, and C. karstii is newly reported to be associated with E. japonicus anthracnose in China. Pathogenicity tests revealed that all tested isolates exhibited pathogenicity in the presence of wounds, emphasizing the need to avoid artificial or mechanical wounds to prevent infection in E. japonicus management. The EC50 values of five fungicides, namely difenoconazole, flusilazole, tebuconazole, hexaconazole, and prochloraz, were found to be less than 10 mg/L, indicating their strong potential for application. Notably, the EC50 of prochloraz was less than 0.05 mg/L for C. theobromicola. These findings offer valuable insights for the management of anthracnose on E. japonicus.

Function Switch of a Fungal Sesterterpene Synthase through Molecular Dynamics Simulation Assisted Alteration of an Aromatic Residue Cluster in the Active Pocket of PfNS.

Terpene synthases (TPSs) play pivotal roles in generating diverse terpenoids through complex cyclization pathways. Protein engineering of TPSs offers a crucial approach to expanding terpene diversity. However, significant potential remains untapped due to limited understanding of the structure-function relationships of TPSs. In this investigation, using a joint approach of molecular dynamics simulations-assisted engineering and site-directed mutagenesis, we manipulated the aromatic residue cluster (ARC) of a bifunctional terpene synthase (BFTPS), Pestalotiopsisfici nigtetraene synthase (PfNS). This led to the discovery of previously unreported catalytic functions yielding different cyclization patterns of sesterterpenes. Specifically, a quadruple variant (F89A/Y113F/W193L/T194W) completely altered PfNS's function, converting it from producing the bicyclic sesterterpene nigtetraene to the tricyclic ophiobolin F. Additionally, analysis of catalytic profiles by double, triple, and quadruple variants demonstrated that the ARC functions as a switch, unprecedently redirecting the production of 5/11 bicyclic (Type B) sesterterpenes to 5/15 bicyclic (Type A) ones. Molecular dynamics simulations and theozyme calculations further elucidated that, in addition to cation-π interactions, C-H∙∙∙π interactions also play a key role in the cyclization patterns. This study offers a feasible strategy in protein engineering of TPSs for various industrial applications.

Identification and genetic analysis of a pervasive 'needle-eye' sperm phenotype in Drosophila sterile hybrid males.

Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila. Hybrid sterility is more often found in the heterogametic (XY or ZW) sex, a trend called Haldane's rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call 'needle-eye sperm', that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used 10 generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye (NE) sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after 10 generations of backcrossing, and only a single region of the X chromosome was associated with sterility in one direction of backcross. Owing to the common phenotype among sterile male hybrids, and the strong effect of individual loci, further exploration of these findings may identify a universal mechanism for the evolution of hybrid sterility.

Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight and pre-mature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin and Lipu, Guangxi Province, and Meishan city, Abe Tibetan and Qiang autonomous prefecture of Sichuan Province. Characteristics of mycelia on PDA, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], ß-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%) and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.

Genome Mining of Fungal Unique Trichodiene Synthase-like Sesquiterpene Synthases.

Sesquiterpenoids served as an important source for natural product drug discovery. Although genome mining approaches have revealed numerous novel sesquiterpenoids and biosynthetic enzymes, the comprehensive landscape of fungal sesquiterpene synthases (STSs) remains elusive. In this study, 123 previously reported fungal STSs were subjected to phylogenetic analysis, resulting in the identification of a fungi-specific STS family known as trichodiene synthase-like sesquiterpene synthases (TDTSs). Subsequently, the application of hidden Markov models allowed the discovery of 517 TDTSs from our in-house fungi genome library of over 400 sequenced genomes, and these TDTSs were defined into 79 families based on a sequence similarity network. Based on the novelty of protein sequences and the completeness of their biosynthetic gene clusters, 23 TDTS genes were selected for heterologous expression in Aspergillus oryzae. In total, 10 TDTSs were active and collectively produced 12 mono- and sesquiterpenes, resulting in the identification of the first chamipinene synthase, as well as the first fungi-derived cedrene, sabinene, and camphene synthases. Additionally, with the guidance of functionally characterized TDTSs, we found that TDTSs in Family 1 could produce bridged-cyclic sesquiterpenes, while those in Family 2 could synthesize spiro- and bridged-cyclic sesquiterpenes. Our research presents a new avenue for the genome mining of fungal sesquiterpenoids.

Isolation, characterization and therapeutic evaluation of a new Acinetobacter virus Abgy202141 lysing Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that easily resists currently available antibiotics. Phages are considered alternative therapeutic agents to conventional antibiotics for the treatment of multidrug-resistant bacteria. We isolated an Acinetobacter virus Abgy202141 from underground sewage in a residential area of Guiyang City in China. Transmission electron microscopy (TEM) analysis showed that Acinetobacter virus Abgy202141 has an icosahedral head attached to a tail. This phage infects A. baumannii strain GY-4, and was found to have a short latent period of 5 min and with a burst size of 189 particles per infected host cell. Additionally, Acinetobacter virus Abgy202141 remained stable at different concentrations of chloroform and varying pH levels and temperatures. Based on SDS-PAGE analysis, it contained 14 proteins with molecular weights ranging from 12 to 125 kDa. The double-strand (ds) DNA genome of Acinetobacter virus Abgy202141 consisted of 41,242 bp with a GC content of 39.4%. It contained 50 open reading frames (ORFs), of which 29 ORFs had identified functions, but no virulence-related genes, antibiotic-resistance genes, or tRNAs were found. Phylogenetic analysis indicated that Acinetobacter virus Abgy202141 was a new phage in the Friunavirus genus. Acinetobacter virus Abgy202141 also showed the ability to prevent A. baumannii infections in the Galleria mellonella in vivo model.

Fruit Anthracnose on Cavendish bananas Caused by Colletotrichum fructicola in Guangxi, China.

Cavendish banana (Musa spp. AAA group) is one of the main fruit crops worldwide. It is widely planted in Guangdong, Hainan, Guangxi, Fujian and Yunnan provinces in southern China. In November 2020, banana fruits with anthracnose symptoms were collected from Dayu Town (N 23.17°, E 109.80°), Guigang City, and Chengjun Town (N 22.60°, E 110.00°), Yulin City, Guangxi Province, China, where the disease was found on about 70% of the banana plants, and on individual fruit, up to 10% of the surface was covered with symptoms. The symptoms initially began with rust-colored spots on the surface of the immature fruit, which gradually became sunken and cracked as the disease progressed. Small tissues (5×5 mm) from the pericarp at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% sodium hypochlorite (NaClO) for 1 min, and washed three times in sterile water. Tissue pieces were placed on potato dextrose ager (PDA) and incubated at 25°C. Fifty-nine morphologically similar colonies were obtained after 5 days of incubation, with 100% isolation frequency. Of 59 isolates, GG1-3 isolated from Guigang City and YL4-2 isolated from Yulin City were selected as representative strains for intensive study. Mycelia were off-white for both isolates and conidia obtained from PDA were cylindrical, unicellular, hyaline and obtuse ends, with sizes of 11.5 ± 1.8×3.9 ± 0.8 µm (n=60) and 11.5 ± 1.6×4.1 ± 0.6 µm (n=60) for GG1-3 and YL4-2, respectively (Prihastuti et al. 2009). Genomic DNA was extracted from 7-day-old aerial mycelia using a DNAsecure Plant Kit (Tiangen Biotech, China). The internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 (ApMAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (White et al. 1990; Silva et al.2012; Templeton et al. 1992). Sequences were deposited in GenBank (ITS, OR596961 to OR596962; GAPDH, OR661771 to OR661772; APMAT, OR661773 to OR661774) and showed 100% identities with the corresponding type strains sequences of C. fructicola. Phylogenetic tree was constructed with software raxmlGUI v.2.0.0 based on sequences of multiple loci (ITS, GAPDH and ApMAT) and Maximum Likelihood method. Phylogenetic analysis revealed that the two isolates and C. fructicola were clustered in the same clade, with 94% bootstrap support. According to morphology and phylogenetic analysis, the two isolates GG1-3 and YL4-2 were identified as C. fructicola. For pathogenicity tests, healthy fruits were surface sterilized with 75% ethanol followed by a wash with sterilized water. Five adjacent needle punctures in a 5-mm-diameter circle were made with a sterilized needle on healthy fruits, followed by inoculation with 20 µL of conidial suspension (106 spores/ml), and sterilized water was used as controls. All banana fruit were incubated in a humid chamber at 28°C. After 4 days, all inoculated fruits showed visible symptoms and had rust-colored spots on the margins, while control banana fruits remained symptomless. The fungus was isolated from the inoculated fruit and the isolates were found to match the morphological and molecular characteristics of the original isolates, confirming Koch's hypothesis. To our knowledge, this is the first report of fruit anthracnose on Cavendish bananas caused by C. fructicola in China. This study will provide valuable information for prevention and management of anthracnose on banana fruit.

Expanding catalytic promiscuity of a bifunctional terpene synthase through a single mutation-induced change in hydrogen-bond network within the catalytic pocket.

Fungal bifunctional terpene synthases (BFTSs) catalyze the formation of numerous di-/sester-/tri-terpenes skeletons. However, the mechanism in controlling the cyclization pattern of terpene scaffolds is rarely deciphered for further application of tuning the catalytic promiscuity of terpene synthases for expanding the chemical space. In this study, we expanded the catalytic promiscuity of Fusarium oxysporum fusoxypene synthase (FoFS) by a single mutation at L89, leading to the production of three new sesterterpenes. Further computational analysis revealed that the reconstitution of the hydrogen-bond (H-bond) network of second-shell residues around the active site of FoFS influences the orientation of the aromatic residue W69 within the first-shell catalytic pocket. Thus, the dynamic orientation of W69 alters the carbocation transport, leading to the production of diverse ring system skeletons. These findings enhance our knowledge on understanding the molecular mechanisms, which could be applied on protein engineering terpene synthases on regulating the terpene skeletons.

Genome sequences of Colletotrichum eleusines and C. echinochloae: two pathogens from Poaceae weeds.

We present two whole-genome sequences of Colletotrichum strains which were isolated from Eleusine indica and Echinochloa crus-galli using Nanopore and Illumina technologies, as part of screening for potential mycoherbicide. The genome sequences will provide important genetic information and will be useful for further research into secondary metabolites of Colletotrichum.

Phenotypic and Genotypic Variation of Cultivated Panax quinquefolius.

American ginseng (Panax quinquefolius) is widely used due to its medicinal properties. Ontario is a major producer of cultivated American ginseng, where seeds were originally collected from the wild without any subsequent scientific selection, and thus the crop is potentially very diverse. A collection of 162 American ginseng plants was harvested from a small area in a commercial garden and phenotyped for morphological traits, such as root grade, stem length, and fresh and dry weights of roots, leaves, stems, and seeds. All of the traits showed a range of values, and correlations were observed between root and stem weights, root dry weight and leaf dry weight, as well as root and leaf fresh weights. The plants were also genotyped using single nucleotide polymorphisms (SNPs) at the PW16 locus. SNP analysis revealed 22 groups based on sequence relatedness with some groups showing no SNPs and others being more diverse. The SNP groups correlated with significant differences in some traits, such as stem length and leaf weight. This study provides insights into the genetic and phenotypic diversity of cultivated American ginseng grown under similar environmental conditions, and the relationship between different phenotypes, as well as genotype and phenotype, will aid in future selection programs to develop American ginseng cultivars with desirable agronomic traits.

Multiple amino acid transporters as carriers load L-valine-phenazine-1-carboxylic acid conjugate into Ricinus sieve tubes for the phloem translocation.

Some transporters play important roles in the uptake and acropetal xylem translocation of vectorized agrochemicals. However, it is poorly understood the basipetally phloem-loading functions of transporters toward vectorized agrochemicals. Here, L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate) uptake was demonstrated carrier-mediated. RcAAP2, RcANT7, and RcLHT1 showed a similarly up-regulated expression pattern from 62 transporter coding genes in Ricinus at 1 h after L-Val or L-Val-PCA treatment. Subcellular localization revealed that fusion RcAAP2-eGFP, RcANT7-eGFP and RcLHT1-eGFP proteins were expressed in the plasma membrane of mesophyll and phloem cells. Yeast assays found that RcAAP2, RcANT7, and RcLHT1 facilitated L-Val-PCA uptake. To further demonstrate the phloem-loading functions, using vacuum infiltration strategy, an Agrobacterium-mediated RNA interference (RNAi) protocol was constructed in seedlings. HPLC detection indicated that L-Val-PCA phloem sap concentrations were significantly decreased 54.5 %, 27.6 %, and 41.6 % after silencing for 72 h and increased 48.3 %, 52.6 %, and 52.4 % after overexpression, respectively. In conclusion, the plasma membrane-located RcAAP2, RcANT7, and RcLHT1 can loaded L-Val-PCA into Ricinus sieve tubes for the phloem translocation, which may aid in the utilization of transporters and molecular design of phloem-mobile fungicides target root or vascular pathogens.

RNA interference of Aspergillus flavus in response to Aspergillus flavus partitivirus 1 infection.

RNA interference (RNAi) is one of the important defense responses against viral infection, but its mechanism and impact remain unclear in mycovirus infections. In our study, reverse genetics and virus-derived small RNA sequencing were used to show the antiviral responses of RNAi components in Aspergillus flavus infected with Aspergillus flavus partitivirus 1 (AfPV1). qRT-PCR revealed that AfPV1 infection induced the expression of the RNAi components in A. flavus compared with noninfected A. flavus. Knock mutants of each RNAi component were generated, but the mutants did not exhibit any obvious phenotypic changes compared with the A. flavus parental strain. However, after AfPV1 inoculation, production of AfPV1 was significantly less than in the parental strain. Furthermore, sporulation was greater in each AfPV1-infected mutant compared with the AfPV1-infected parental A. flavus. We also investigated the sensitivity of virus-free and AfPV1-infected RNAi mutants and the parental strain to cell wall stress, osmotic stress, genotoxic stress, and oxidative stress. The mutants of DCLs and AGOs infected by AfPV1 displayed more changes than RDRP mutants in response to the first three stresses. Small RNA sequencing analysis suggested that AfPV1 infection reduced the number of unique reads of sRNA in A. flavus, although there were many vsiRNA derived from the AfPV1 genome. GO term and KEGG pathway analyses revealed that the functions of sRNA affected by AfPV1 infection were closely related to vacuole production. These results provide a better understanding of the functional role of RNAi in the impact of AfPV1 on the hypovirulence of A. flavus.

A journey with psychedelic mushrooms: From historical relevance to biology, cultivation, medicinal uses, biotechnology, and beyond.

Psychedelic mushrooms containing psilocybin and related tryptamines have long been used for ethnomycological purposes, but emerging evidence points to the potential therapeutic value of these mushrooms to address modern neurological, psychiatric health, and related disorders. As a result, psilocybin containing mushrooms represent a re-emerging frontier for mycological, biochemical, neuroscience, and pharmacology research. This work presents crucial information related to traditional use of psychedelic mushrooms, as well as research trends and knowledge gaps related to their diversity and distribution, technologies for quantification of tryptamines and other tryptophan-derived metabolites, as well as biosynthetic mechanisms for their production within mushrooms. In addition, we explore the current state of knowledge for how psilocybin and related tryptamines are metabolized in humans and their pharmacological effects, including beneficial and hazardous human health implications. Finally, we describe opportunities and challenges for investigating the production of psychedelic mushrooms and metabolic engineering approaches to alter secondary metabolite profiles using biotechnology integrated with machine learning. Ultimately, this critical review of all aspects related to psychedelic mushrooms represents a roadmap for future research efforts that will pave the way to new applications and refined protocols.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Forest Type and Climate Outweigh Soil Bank in Shaping Dynamic Changes in Macrofungal Diversity in the Ancient Tree Park of Northeast China.

The community structure of macrofungi is influenced by multiple complex factors, including climate, soil, vegetation, and human activities, making it challenging to discern their individual contributions. To investigate the dynamic changes in macrofungal diversity in an Ancient Tree Park located in Northeast China and explore the factors influencing this change, we collected 1007 macrofungi specimens from different habitats within the park and identified 210 distinct fungal species using morphological characteristics and ITS sequencing. The species were classified into 2 phyla, 6 classes, 18 orders, 55 families, and 94 genera. We found macrofungal compositions among different forest types, with the mixed forest displaying the highest richness and diversity. Climatic factors, particularly rainfall and temperature, positively influenced macrofungal species richness and abundance. Additionally, by analyzing the soil fungal community structure and comparing aboveground macrofungi with soil fungi in this small-scale survey, we found that the soil fungal bank is not the main factor leading to changes in the macrofungal community structure, as compared to the influence of climate factors and forest types. Our findings provide valuable insights into the dynamic nature of macrofungal diversity in the Ancient Tree Park, highlighting the influence of climate and forest type.

Complete genome sequence of a novel chrysovirus infecting Aspergillus terreus.

A double-stranded RNA (dsRNA) mycovirus was obtained from Aspergillus terreus strain HJ3-26 and designated "Aspergillus terreus chrysovirus 1" (AtCV1). It consists of four dsRNA segments (dsRNA1-4) with lengths of 3612 bp, 3132 bp, 3153 bp, and 3144 bp, respectively. Sequence analysis showed that dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA2 encodes a capsid protein, and both dsRNA3 and dsRNA4 encode hypothetical proteins. Phylogenetic analysis of the RdRp suggested that AtCV1 is a member of a new species of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus obtained from A. terreus.

Amino acid permease RcAAP1 increases the uptake and phloem translocation of an L-valine-phenazine-1-carboxylic acid conjugate.

Amino acid conjugates of pesticides can promote the phloem translocation of parent ingredients, allowing for the reduction of usage, and decreased environmental pollution. Plant transporters play important roles in the uptake and phloem translocation of such amino acid-pesticide conjugates such as L-Val-PCA (L-valine-phenazine-1-carboxylic acid conjugate). However, the effects of an amino acid permease, RcAAP1, on the uptake and phloem mobility of L-Val-PCA are still unclear. Here, the relative expression levels of RcAAP1 were found to be up-regulated 2.7-fold and 2.2-fold by the qRT-PCR after L-Val-PCA treatments of Ricinus cotyledons for 1 h and 3 h, respectively. Subsequently, expression of RcAAP1 in yeast cells increased the L-Val-PCA uptake (0.36 µmol/107 cells), which was 2.1-fold higher than the control (0.17 µmol/107 cells). Pfam analysis suggested RcAAP1 with its 11 transmembrane domains belongs to the amino acid transporter family. Phylogenetic analysis found RcAAP1 to be strongly similar to AAP3 in nine other species. Subcellular localization showed that fusion RcAAP1-eGFP proteins were observed in the plasma membrane of mesophyll cells and phloem cells. Furthermore, overexpression of RcAAP1 for 72 h significantly increased the phloem mobility of L-Val-PCA in Ricinus seedlings, and phloem sap concentration of the conjugate was 1.8-fold higher than the control. Our study suggested that RcAAP1 as carrier was involved in the uptake and phloem translocation of L-Val-PCA, which could lay foundation for the utilization of amino acids and further development of vectorized agrochemicals.

The evolution of mini-chromosomes in the fungal genus Colletotrichum.

Anthracnose diseases caused by Colletotrichum species are among the most common fungal diseases. These symptoms typically manifest as dark, sunken lesions on leaves, stems, and fruit. In China, mango anthracnose seriously affects fruit yield and quality. Genome sequencing of several species shows the presence of mini-chromosomes. These are thought to contribute to virulence, but their formation and activity remain to be fully elucidated. Here, we assembled 17 Colletotrichum genomes (16 isolated from mango plus one from persimmon) through PacBio long-read sequencing. Half of the assembled scaffolds had telomeric repeats at both ends indicating full-length chromosomes. Based on comparative genomics analysis at interspecies and intraspecies levels, we identified extensive chromosomal rearrangements events. We analyzed mini-chromosomes of Colletotrichum spp. and found large variation among close relatives. In C. fructicola, homology between core chromosomes and mini-chromosomes suggested that some mini-chromosomes were generated by recombination of core chromosomes. In C. musae GZ23-3, we found 26 horizontally transferred genes arranged in clusters on mini-chromosomes. In C. asianum FJ11-1, several potential pathogenesis-related genes on mini-chromosomes were upregulated, especially in strains with highly pathogenic phenotypes. Mutants of these upregulated genes showed obvious defects in virulence. Our findings provide insights into the evolution and potential relationships to virulence associated with mini-chromosomes. IMPORTANCE Colletotrichum is a cosmopolitan fungal genus that seriously affects fruit yield and quality of many plant species. Mini-chromosomes have been found to be related to virulence in Colletotrichum. Further examination of mini-chromosomes can help us elucidate some pathogenic mechanisms of Colletotrichum. In this study, we generated novel assemblies of several Colletotrichum strains. Comparative genomic analyses within and between Colletotrichum species were conducted. We then identified mini-chromosomes in our sequenced strains systematically. The characteristics and generation of mini-chromosomes were investigated. Transcriptome analysis and gene knockout revealed pathogenesis-related genes located on mini-chromosomes of C. asianum FJ11-1. This study represents the most comprehensive investigation of chromosome evolution and potential pathogenicity of mini-chromosomes in the Colletotrichum genus.

Identification and characterization of extracellular GH3 ß-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) ß-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its ß-glucosidase has not been identified. In this study, a GH3 ß-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl ß-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on ß-glucobioses with ß1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to ß1-6 linkage. MnBG3A has similar features to those of ß-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.