Natural variation at OsCERK1 regulates arbuscular mycorrhizal symbiosis in rice.

The symbiotic interaction between arbuscular mycorrhizal fungi (AMF) and land plants is essential for efficient nutrient acquisition and utilisation. Our understanding of key processes controlling the AMF colonisation in rice is still limited. Dongxiang wild rice (DY) exhibited a stronger colonisation with Rhizophagus irregularis than the rice cultivar Zhongzao 35 (ZZ35). Chromosome segment substitution lines were constructed and the OsCERK1 gene from DY was mapped. Transgenic plants in the japonica rice Zhonghua 11 (ZZ11) were constructed to compare root colonisation by AMF. Chromosome single-segment substitution lines containing OsCERK1DY showed higher phosphorus content and grain yield relative to ZZ35. Four amino acids substitutions were identified among the OsCERK1 haplotypes of DY, ZZ35 and ZH11 and two of these were in the second lysine-motif domain, which is essential for the differences of AMF colonisation level among rice varieties. Heterologous expression of OsCERK1DY in ZH11 significantly enhanced AMF colonisation and increased resistance against the pathogenic fungi Magnaporthe oryzae. Notably, the OsCERK1DY haplotype was absent from 4660 cultivated rice varieties. We conclude that OsCERK1 is a key gene affecting the symbiotic interaction with AMF and OsCERK1DY has the biotechnological potential to increase rice phosphorus acquisition and utilisation efficiency for sustainable agriculture.

Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9.

Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.

Streptomyces alfalfae sp. nov. and comparisons with its closest taxa Streptomyces silaceus, Streptomyces flavofungini and Streptomyces intermedius.

A novel streptomycete strain, designated XY25T, was isolated from the rhizosphere soil in an alfalfa field in Jingyang, Shanxi, China. The isolate showed optimal growth at 37 °C, and was capable of growing at pH 6-10 and in the presence of 0-6 % (w/v) NaCl. Mycelia of strain XY25T appeared spiral and developed into white spore chains with long-rod spores and a smooth surface. The 16S rRNA gene sequence of XY25T was determined and was found to be highly similar to those of species of the genus Streptomyces including Streptomyces silaceus DSM 41861T (99.11 % 16S rRNA gene sequence similarity), Streptomyces flavofungini DSM 40366T (98.49 %) and Streptomyces intermedius DSM 40372T (98.43 %), all of which were used for further characterization. Each of the four streptomycetes showed distinctive patterns of carbon usage and fatty acids composition. Analysis of cellular components of strain XY25T revealed ll-diaminopimelic acid as diagnostic diamino acid and xylose as the major sugar, whereas polar lipids were determined as phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, an unknown phospholipid, two unknown phosphatidylinositol mannosides and several unknown lipids. Menaquinones were dominated by MK-9(H6) and MK-9(H8), and the main fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. DNA-DNA hybridization studies indicated that strain XY25T showed relatedness values of 35.2-40.42 % with the closest related species. Based on these results, strain XY25T represents a novel species of the genus Streptomyces, for which the name Streptomyces alfalfae sp. nov. is proposed. The type strain is XY25T ( = KCTC 39571T = CCTCC AA2015019T).

Exosome Component 4 Promotes Epithelial Ovarian Cancer Cell Proliferation, Migration, and Invasion via the Wnt Pathway.

BACKGROUND: Of gynecologic malignancies, ovarian cancer is the leading cause of death, mainly due to the lack of sensitive tumor markers, which means it almost always presents at an advanced stage. Exosome Component 4 (EXOSC4) is involved in RNA degradation, but its role in epithelial ovarian cancer (EOC) is unclear. METHODS: The expression levels of EXOSC4 in EOC and normal ovarian tissue specimens were determined by immunohistochemical staining. The overall survival (OS) and progression-free survival (PFS) of patients with EOC were evaluated after patients were classified into high and low EXOSC4 expression groups, and the Cox regression model was established to identify independent predictors of patient prognosis. The effects of EXOSC4 on proliferation, colony formation, migration, and invasion were examined in the SKOV-3 and HO8910 cell lines by lentivirus-mediated shRNA knockdown. Flow cytometry was used to detect cell cycle changes. The mRNA levels of cyclin D1, CDK4, and c-myc were detected by RT-PCR. The protein expression levels of ß-catenin, cyclin D1, CDK4, c-myc, vimentin, N-cadherin, and E-cadherin were assessed by western blot. Wnt/ß-catenin activation was measured by TCF/LEF reporter assay. RESULTS: EXOSC4 was significantly elevated in EOC tissues and cell lines. High EXOSC4 expression was correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage and pathological grade, and identified as an independent predictor of shorter OS and PFS. EXOSC4 knockdown suppressed proliferation, migration, and invasion in EOC cell lines. Cells were arrested at G0/G1 phase after EXOSC4 knockdown. The mRNA levels of cyclin D1, CDK4, and c-myc were decreased. ß-catenin, cyclin D1, CDK4, c-myc, vimentin, and N-cadherin protein expression levels were reduced, while those of E-cadherin was increased. Wnt/ß-catenin activity was suppressed after the EXOSC4 knockdown. CONCLUSIONS: EXOSC4 is involved in EOC. Knockdown of EXOSC4 can inhibit the proliferation, migration, and invasion ability of EOC by suppressing the Wnt pathway. EXOSC4 is expected to be a novel biomarker and molecular target in EOC.

Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis.

Arbuscular mycorrhizal (AM) fungi are soil-borne fungi belonging to the ancient phylum Glomeromycota and are important symbionts of the arbuscular mycorrhiza, enhancing plant nutrient acquisition and resistance to various abiotic stresses. In contrast to their significant physiological implications, the molecular basis involved is poorly understood, largely due to their obligate biotrophism and complicated genetics. Here, we identify and characterize three genes termed Fm201, Ri14-3-3 and RiBMH2 that encode 14-3-3-like proteins in the AM fungi Funneliformis mosseae and Rhizophagus irregularis, respectively. The transcriptional levels of Fm201, Ri14-3-3 and RiBMH2 are strongly induced in the pre-symbiotic and symbiotic phases, including germinating spores, intraradical hyphae- and arbuscules-enriched roots. To functionally characterize the Fm201, Ri14-3-3 and RiBMH2 genes, we took advantage of a yeast heterologous system owing to the lack of AM fungal transformation systems. Our data suggest that all three genes can restore the lethal Saccharomyces cerevisiae bmh1 bmh2 double mutant on galactose-containing media. Importantly, yeast one-hybrid analysis suggests that the transcription factor RiMsn2 is able to recognize the STRE (CCCCT/AGGGG) element present in the promoter region of Fm201 gene. More importantly, Host-Induced Gene Silencing of both Ri14-3-3 and RiBMH2 in Rhizophagus irregularis impairs the arbuscule formation in AM symbiosis and inhibits the expression of symbiotic PT4 and MST2 genes from plant and fungal partners, respectively. We further subjected the AM fungus-Medicago truncatula association system to drought or salinity stress. Accordingly, the expression profiles in both mycorrhizal roots and extraradical hyphae reveal that these three 14-3-3-like genes are involved in response to drought or salinity stress. Collectively, our results provide new insights into molecular functions of the AM fungal 14-3-3 proteins in abiotic stress responses and arbuscule formation during AM symbiosis.

Arbuscular Mycorrhizal Symbiosis Requires a Phosphate Transceptor in the Gigaspora margarita Fungal Symbiont.

The majority of terrestrial vascular plants are capable of forming mutualistic associations with obligate biotrophic arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycota. This mutualistic symbiosis provides carbohydrates to the fungus, and reciprocally improves plant phosphate uptake. AM fungal transporters can acquire phosphate from the soil through the hyphal networks. Nevertheless, the precise functions of AM fungal phosphate transporters, and whether they act as sensors or as nutrient transporters, in fungal signal transduction remain unclear. Here, we report a high-affinity phosphate transporter GigmPT from Gigaspora margarita that is required for AM symbiosis. Host-induced gene silencing of GigmPT hampers the development of G. margarita during AM symbiosis. Most importantly, GigmPT functions as a phosphate transceptor in G. margarita regarding the activation of the phosphate signaling pathway as well as the protein kinase A signaling cascade. Using the substituted-cysteine accessibility method, we identified residues A146 (in transmembrane domain [TMD] IV) and Val357 (in TMD VIII) of GigmPT, both of which are critical for phosphate signaling and transport in yeast during growth induction. Collectively, our results provide significant insights into the molecular functions of a phosphate transceptor from the AM fungus G. margarita.

HDAC9 promotes glioblastoma growth via TAZ-mediated EGFR pathway activation.

Histone deacetylase 9 (HDAC9), a member of class II HDACs, regulates a wide variety of normal and abnormal physiological functions. We found that HDAC9 is over-expressed in prognostically poor glioblastoma patients. Knockdown HDAC9 decreased proliferation in vitro and tumor formation in vivo. HDAC9 accelerated cell cycle in part by potentiating the EGFR signaling pathway. Also, HDAC9 interacted with TAZ, a key downstream effector of Hippo pathway. Knockdown of HDAC9 decreased the expression of TAZ. We found that overexpressed TAZ in HDAC9-knockdown cells abrogated the effects induced by HDAC9 silencing both in vitro and in vivo. We demonstrated that HDAC9 promotes tumor formation of glioblastoma via TAZ-mediated EGFR pathway activation, and provide the evidence for promising target for the treatment of glioblastoma.

Characterization and identification of the integrin family in silkworm, Bombyx mori.

As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five ß subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The ß subunits mainly gather in the insect ßν group except the ß1 subunit which belongs to the insect ß group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and ß2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins.