Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

Transcriptome analysis reveals the regulatory mode by which NAA promotes the growth of Armillaria gallica.

A symbiotic relationship is observed between Armillaria and the Chinese herbal medicine Gastrodia elata (G. elata). Armillaria is a nutrient source for the growth of G. elata, and its nutrient metabolism efficiency affects the growth and development of G. elata. Auxin has been reported to stimulate Armillaria species, but the molecular mechanism remains unknown. We found that naphthalene acetic acid (NAA) can also promote the growth of A. gallica. Moreover, we identified a total of 2071 differentially expressed genes (DEGs) by analyzing the transcriptome sequencing data of A. gallica at 5 and 10 hour of NAA treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these unigenes were significantly enriched in the metabolism pathways of arginine, proline, propanoate, phenylalanine and tryptophan. The expression levels of the general amino acid permease (Gap), ammonium transporter (AMT), glutamate dehydrogenase (GDH), glutamine synthetase (GS), Zn(II) 2Cys6 and C2H2 transcription factor genes were upregulated. Our transcriptome analysis showed that the amino acid and nitrogen metabolism pathways in Armillaria were rapidly induced within hours after NAA treatment. These results provide valuable insights into the molecular mechanisms by which NAA promotes the growth of Armillaria species.

De novo RNA sequencing and analysis reveal the putative genes involved in diterpenoid biosynthesis in Aconitum vilmorinianum roots.

In this study, the putative genes involved in diterpenoid alkaloids biosynthesis in A. vilmorinianum roots were revealed by transcriptome sequencing. 59.39 GB of clean bases and 119,660 unigenes were assembled, of which 69,978 unigenes (58.48%) were annotated. We identified 27 classes of genes (139 candidate genes) involved in the synthesis of diterpenoid alkaloids, including the mevalonate (MVA) pathway, the methylerythritol 4-phosphate (MEP) pathway, the farnesyl diphosphate regulatory pathway, and the diterpenoid scaffold synthetic pathway. 12 CYP450 genes were identified. We found that hydroxymethylglutaryl-CoA reductase was the key enzyme in MVA metabolism, which was regulated by miR6300. Transcription factors, such as bHLH, AP2/EREBP, and MYB, used to synthesize the diterpenes were analyzed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02646-6.

[Molecular cloning, bacterial expression analysis and functional characterization of pathogenesis-related PR10-2 gene in Panax notoginseng].

Base on the transcriptome analysis and RT-PCR techniques,a pathogenesis-related protein 10 gene was isolated from Panax notoginseng root and named as PnPR10-2. Bioinformatics and phylogenetic trees analysis revealed that open reading frame (ORF) of PnPR10-2 was 465 bp in length,encoding 154 amino acids,containing one typical conserved domain of pathogenesis related protein Bet v I family, and showed high similarity with that from P. ginseng. The recombinant expressed plasmid pET32a(+)-PnPR10-2 was expressed in Escherichia coli BL21. The expression conditions were optimized and it could be expressed well in soluble and inclusion body protein. Purified PnPR10-2 recombinant protein from the supernatant of cells was used to analysis the pathogen resistance activity by paper method. The purified recombinant protein could inhibit typical root rot disease pathogen (Fusarium solani and Cylindrocarpon destructans）growth evidently, we conjecture that PnPR10-2 may participated in defense response of P. notoginseng resistance to root rot disease pathogen.

Estimation of nitrogen and phosphorus flows in livestock production in Dianchi Lake basin, China.

We assessed the nitrogen (N) and phosphorus (P) flows in intensified livestock production systems by investigating nutrient budgets and cycling in the basin of Dianchi Lake, one of the most eutrophic lakes in China. We conducted field surveys based on feed samplings and interviews of livestock farmers. The N and P in local and external feeds, animal body retentions, animal products and excretions were calculated at the individual level for dairy cattle, fattening pigs, breeding sows, broilers and laying hens. The N and P flows in the total livestock production system in the area were estimated by multiplying the individual N and P budgets by the number of animals. For the dairy and fattening pig productions, N and P supplied from local crops or by-products accounted for large parts of the inputs. For the other livestock categories, most of the N and P inputs depended on external resources. The N and P outputs through animal manure into the cropland were 287 and 66 kg/ha/year, respectively, which were higher than the N and P inputs into the livestock production systems from the cropland. The N and P loads from manure should be reduced for the establishment of sustainable agricultural production systems.

Irrigation system and land use effect on surface water quality in river, at Lake Dianchi, Yunnan, China.

The surface water samples were collected in river Dahe and its tributaries, which flow into severely eutrophic lake Dianchi, Yunnan Province, China, in order to elucidate factors controlling water quality fluctuations. The temporal and spatial distribution of water quality tendency was observed. The water quality of each river is dependent on the hydrology effect such water gate and circulating irrigation system. We must consider the hydrology effect to accurately understand water quality variations of river in this study field. In river without highly circulating irrigation system or water gate effect, the downstream nitrate nitrogen (NO3-N) concentration increase occurred in area dominated by open field cultivation, whereas the NO3-N concentration was constant or decreased in area dominated by greenhouse land use. This result suggests that greenhouse covers the soil from precipitation, and nitrate load of greenhouse could be less than that of open field cultivation while the rainfall event. In the upper reaches of river, where is dominated by open field cultivation, there were no sharp increase dissolved molybdate reactive phosphorus and total phosphorus concentration, but P load was accumulated in the lower reaches of river, whose predominant land use is greenhouse. Although the P sources is unclear in this study, greenhouse area may have potential of P loads due to its high P content in greenhouse soil. Considering hydrology effect is necessary to determine what the major factor is influencing the water quality variation, especially in area with highly complicated irrigation system in this studying site.

Spirocyclic acylphloroglucinol derivatives from Hypericum beanii.

Four new acylphloroglucinols with an unusual 6/6/5 spirocyclic skeleton, hyperbeanols A-D (1-4), were isolated from the methanol extract of Hypericum beanii along with 16 known compounds. Their structures were established on the basis of spectroscopic and X-ray diffraction analysis. Hyperbeanols A-C were three stereoisomers different only at the relative configuration of C-4 and C-13, which were distinguished by the nuclear Overhauser effect spectroscopy (NOESY) spectroscopic data in combination with the single X-ray analysis of hyperbeanol A (1). The cytotoxic activity of hyperbeanols A-D against the cancer cell lines SK-BR-3, HL-60, SMMC-7721, PANC-1, MCF-7, and K562 was also evaluated.

Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C4-like pathway is proposed.