Genomic Characterization Provides New Insights Into the Biosynthesis of the Secondary Metabolite Huperzine a in the Endophyte Colletotrichum gloeosporioides Cg01

A reliable source of Huperzine A (HupA) meets an urgent need due to its wide use in Alzheimer's disease treatment. In this study, we sequenced and characterized the whole genomes of two HupA-producing endophytes, Penicillium polonicum hy4 and Colletotrichum gloeosporioides Cg01, to clarify the mechanism of HupA biosynthesis. The whole genomes of hy4 and Cg01 were 33.92 and 55.77 Mb, respectively. We compared the differentially expressed genes (DEGs) between the induced group (with added extracts of Huperzia serrata) and a control group. We focused on DEGs with similar expression patterns in hy4 and Cg01. The DEGs identified in GO (Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were primarily located in carbon and nitrogen metabolism and nucleolus, ribosome, and rRNA processing. Furthermore, we analyzed the gene expression for HupA biosynthesis genes proposed in plants, which include lysine decarboxylase (LDC), copper amine oxidase (CAO), polyketides synthases (PKS), etc. Two LDCs, one CAO, and three PKSs in Cg01 were selected as prime candidates for further validation. We found that single candidate biosynthesis-gene knock-out did not influence the HupA production, while both LDC gene knock-out led to increased HupA production. These results reveal that HupA biosynthesis in endophytes might differ from that proposed in plants, and imply that the HupA-biosynthesis genes in endophytic fungi might co-evolve with the plant machinery rather than being acquired through horizontal gene transfer (HGT). Moreover, we analyzed the function of the differentially expressed epigenetic modification genes. HupA production of the histone acetyltransferase (HAT) deletion mutant ΔCgSAS-2 was not changed, while that of the histone methyltransferase (HMT) and histone deacetylase (HDAC) deletion mutants ΔCgClr4, ΔCgClr3, and ΔCgSir2-6 was reduced. Recovery of HupA-biosynthetic ability can be achieved by retro-complementation, demonstrating that HMT and HDACs associated with histone modification are involved in the regulation of HupA biosynthesis in endophytic fungi. This is the first report on epigenetic modification in high value secondary metabolite- producing endophytes. These findings shed new light on HupA biosynthesis and regulation in HupA-producing endophytes and are crucial for industrial production of HupA from fungi

Genome and secretome analysis of Pochonia chlamydosporia provide new insight into egg-parasitic mechanisms

Pochonia chlamydosporia infects eggs and females of economically important plant-parasitic nematodes. The fungal isolates parasitizing different nematodes are genetically distinct. To understand their intraspecific genetic differentiation, parasitic mechanisms, and adaptive evolution, we assembled seven putative chromosomes of P. chlamydosporia strain 170 isolated from root-knot nematode eggs (~44 Mb, including 7.19% of transposable elements) and compared them with the genome of the strain 123 (~41 Mb) isolated from cereal cyst nematode. We focus on secretomes of the fungus, which play important roles in pathogenicity and fungus-host/environment interactions, and identified 1,750 secreted proteins, with a high proportion of carboxypeptidases, subtilisins, and chitinases. We analyzed the phylogenies of these genes and predicted new pathogenic molecules. By comparative transcriptome analysis, we found that secreted proteins involved in responses to nutrient stress are mainly comprised of proteases and glycoside hydrolases. Moreover, 32 secreted proteins undergoing positive selection and 71 duplicated gene pairs encoding secreted proteins are identified. Two duplicated pairs encoding secreted glycosyl hydrolases (GH30), which may be related to fungal endophytic process and lost in many insect-pathogenic fungi but exist in nematophagous fungi, are putatively acquired from bacteria by horizontal gene transfer. The results help understanding genetic origins and evolution of parasitism-related genes.This work was supported by the National Key Research and Development (R&D) Plan of China (2016YFC1201100), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS)

The Novel Secreted Meloidogyne incognita Effector MiISE6 Targets the Host Nucleus and Facilitates Parasitism in Arabidopsis

Meloidogyne incognita is highly specialized parasite that interacts with host plants using a range of strategies. The effectors are synthesized in the esophageal glands and secreted into plant cells through a needle-like stylet during parasitism. In this study, based on RNA-seq and bioinformatics analysis, we predicted 110 putative Meloidogyne incognita effectors that contain nuclear localization signals (NLSs). Combining the Burkholderia glumae–pEDV based screening system with subcellular localization, from 20 randomly selected NLS effector candidates, we identified an effector MiISE6 that can effectively suppress B. glumae-induced cell death in Nicotiana benthamiana, targets to the nuclei of plant cells, and is highly expressed in early parasitic J2 stage. Sequence analysis showed that MiISE6 is a 157-amino acid peptide, with an OGFr_N domain and two NLS motifs. Hybridization in situ verified that MiISE6 is expressed in the subventral esophageal glands. Yeast invertase secretion assay validated the function of the signal peptide harbored in MiISE6. Transgenic Arabidopsis thaliana plants expressing MiISE6 become more susceptible to M. incognita. Inversely, the host-derived RNAi of MiISE6 of the nematode can decrease its parasitism on host. Based on transcriptome analysis of the MiISE6 transgenic Arabidopsis samples and the wild-type samples, we obtained 852 differentially expressed genes (DEGs). Integrating Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, we found that expression of MiISE6 in Arabidopsis can suppress jasmonate signaling pathway. In addition, the expression of genes related to cell wall modification and the ubiquitination proteasome pathway also have detectable changes in the transgenic plants. Results from the present study suggest that MiISE6 is involved in interaction between nematode-plant, and plays an important role during the early stages of parasitism by interfering multiple signaling pathways of plant. Moreover, we found homologs of MiISE6 in other sedentary nematodes, Meloidogyne hapla and Globodera pallida. Our experimental results provide evidence to decipher the molecular mechanisms underlying the manipulation of host immune defense responses by plant parasitic nematodes, and transcriptome data also provide useful information for further study nematode–plant interactions

URA3 as a Selectable Marker for Disruption and Functional Assessment of <i>PacC</i> Gene in the Entomopathogenic Fungus <i>Isaria javanica</i>

An effective selection marker is necessary for genetic engineering and functional genomics research in the post-genomic era. Isaria javanica is an important entomopathogenic fungus with a broad host range and prospective biocontrol potentials. Given that no antibiotic marker is available currently in this fungus, developing an effective selection marker is necessary. In this study, by applying overlap PCR and split-marker deletion strategy, combining PEG-mediated protoplasm transformation method, the uridine auxotrophy gene (ura3) in the I. javanica genome was knocked out. Then, using this transformation system, the pH response transcription factor gene (IjpacC) was disrupted successfully. Loss of IjpacC gene results in an obvious decrease in conidial production, but little impact on mycelial growth. The virulence of the ΔIjpacC mutant on caterpillars is similar to that of the wild-type strain. RT-qPCR detection shows that expression level of an acidic-expressed S53 gene (IF1G_06234) in ΔIjpacC mutant is more significantly upregulated than in the wild-type strain during the fungal infection on caterpillars. Our results indicate that a markerless transformation system based upon complementation of uridine auxotrophy is successfully developed in I. javanica, which is useful for exploring gene function and for genetic engineering to enhance biological control potential of the fungus

Ethiopian Mustard (Brassica carinata A. Braun) as an Alternative Energy Source and Sustainable Crop

Energy and food source crop demand claims to be vulnerable to climate change impacts. The new and orphan crops, which in the past have received only limited research attention but are sustainable to environmental systems, are needed. In this review, we summarize the available literature about Ethiopian mustard as an alternative energy source and its sustainable economic importance as a new promising Brassicacea crop for new opportunities in the face of producing sustainable environment and energy development. Ethiopian mustard has many advantages and can be adopted to replace crops that are susceptible to adverse environmental conditions. Ethiopian mustard is becoming a new promising Brassicaceae crop with the current global energy demand increases. However, researchers have only focused on energy source production which has resulted in developing high erucic acid varieties. This results partly in limited studies on developing Ethiopian mustard edible oil varieties. The adoption and scaling-up of this promising crop as an oilseed crop in developing countries and Mediterranean conditions can sustain the impact of climate change with the demand for food and energy debate concepts. Indeed, further agronomic, quality and genomic studies on oilseed nutritional traits for efficient breeding and utilization are needed

Global Protein–Protein Interaction Network of Rice Sheath Blight Pathogen

<i>Rhizoctonia solani</i> is the major pathogenic fungi of rice sheath blight. It is responsible for the most serious disease of rice (<i>Oryza sativa</i> L.) and causes significant yield losses in rice-growing countries. Identifying the protein–protein interaction (PPI) maps of <i>R. solani</i> can provide insights into the potential pathogenic mechanisms and assign putative functions to unknown genes. Here, we exploited a PPI map of <i>R. solani</i> anastomosis group 1 IA (AG-1 IA) based on the interolog and domain–domain interaction methods. We constructed a core subset of high-confidence protein networks consisting of 6705 interactions among 1773 proteins. The high quality of the network was revealed by comprehensive methods, including yeast two-hybrid experiments. Pathogenic interaction subnetwork, secreted proteins subnetwork, and mitogen-activated protein kinase (MAPK) cascade subnetwork and their interacting partners were constructed and analyzed. Moreover, to exactly predict the pathogenic factors, the expression levels of the interaction proteins were investigated by analyzing RNA sequences that consisted of samples from the entire infection progress. The PPIs offer an exceptionally rich source of data that can be used to understand the gene functions and biological processes of this serious disease at the system level

Selection on BrFLC1 Is Related to Intraspecific Diversity of Brassica rapa Vegetables

Flowering time is important for Brassica rapa vegetables because premature bolting before harvest can lower yield and quality. FLOWERING LOCUS C (FLC) acts as a key repressor of flowering. In this study, we identified a nonsynonymous mutation at the 58th nucleotide of exon1 in BrFLC1 (named as Pe1+58 (A/C)) by screening resequencing data of 199 B. rapa accessions and verified this mutation as being related to flowering time variation. Strong linkage inheritance was detected between this locus and a previously reported splicing site mutation at intron 6 of BrFLC1 (Pi6+1 (G/A)), showing as co-occurrence of BrFLC1Pe1+58(A) and BrFLC1Pi6+1(G), named as haplotype H1: AG, or co-occurrence of BrFLC1Pe1+58(C) and BrFLC1Pi6+1(A), named as haplotype H2: CA. The frequency distribution of BrFLC1 haplotypes skewed to the haplotype H1 in turnip, broccoletto, mizuna, komatsuna, and taicai, while it was skewed to the haplotype H2 in caixin, pak choi, zicaitai, and wutacai. The frequencies of the two haplotypes were comparable in Chinese cabbage. This indicated that BrFLC1 haplotypes were related to B. rapa intraspecific diversification. Further analysis of a Chinese cabbage collection revealed that accessions from the spring ecotype preferred to keep H1: AG and almost all accessions from the summer ecotype were H2: CA. The early flowering haplotype of BrFLC1 was purified in summer Chinese cabbage, indicating that BrFLC1 had been strongly selected during genetic improvement of summer Chinese cabbages. A significant difference in flowering time of F2 individuals with the homologous BrFLC1Pi6+1(G) allele but different BrFLC1Pe1+58 (A/C) alleles, indicated that this locus had independent genetic effects on flowering time. The newly identified allelic diversity of BrFLC1 can be used for breeding of resistance to premature bolting in B. rapa vegetables