Zide Jiang

MicroRNA miR-378 Regulates Nephronectin Expression Modulating Osteoblast Differentiation by Targeting GalNT-7

MicroRNAs (miRNAs) are small fragments of single-stranded RNA containing 18-24 nucleotides, and are generated from endogenous transcripts. MicroRNAs function in post-transcriptional gene silencing by targeting the 3′-untranslated region (UTR) of mRNAs, resulting in translational repression. We have developed a system to study the role of miRNAs in cell differentiation. We have found that one of the miRNAs tested in our system (miR-378, also called miR-378*) plays a role in modulating nephronectin-mediated differentiation in the osteoblastic cell line, MC3T3-E1. Nephronectin is an extracellular matrix protein, and we have demonstrated that its over-expression enhanced osteoblast differentiation and bone nodule formation. Furthermore, we found that the nephronectin 3′-untranslated region (3′UTR) contains a binding site for miR-378. Stable transfection of MC3T3-E1 cells with miR-378 inhibited cell differentiation. MC3T3-E1 cells stably transfected with nephronectin exhibited higher rates of differentiation and nodule formation as compared with cells transfected with nephronectin containing the 3′UTR in the early stages of development, suggesting that endogenous miR-378 is present and active. However, in the later stages of MC3T3-E1 development, the differentiation rates were opposite, with higher rates of differentiation and nodule formation in the cells over-expressing the 3′UTR of nephronectin. This appeared to be the consequence of competition between nephronectin and UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7) for miR-378 binding, resulting in increased GalNT7 activity, which in turn lead to increased nephronectin glycosylation and product secretion, thereby resulting in a higher rate of osteoblast differentiation.

MicroRNA miR-24 enhances tumor invasion and metastasis by targeting PTPN9 and PTPRF to promote EGF signaling

Summary MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

Mitogen-Activated Protein Kinases Are Associated with the Regulation of Physiological Traits and Virulence in Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence.

The mating-type locus b of the sugarcane smut Sporisorium scitamineum is essential for mating, filamentous growth and pathogenicity.

Sporisorium scitamineum is the causal agent of sugarcane smut, which is one of the most serious constraints to global sugarcane production. S. scitamineum and Ustilago maydis are two closely related smut fungi, that are predicted to harbor similar sexual mating processes/system. To elucidate the molecular basis of sexual mating in S. scitamineum, we identified and deleted the ortholog of mating-specific U. maydis locus b, in S. scitamineum. The resultant b-deletion mutant was defective in mating and pathogenicity in S. scitamineum. Furthermore, a functional b locus heterodimer could trigger filamentous growth without mating in S. scitamineum, and functionally replace the b locus in U. maydis in terms of triggering aerial filament production and forming solopathogenic strains, which do not require sexual mating prior to pathogenicity on the host plants.

Sequencing of the Litchi Downy Blight Pathogen Reveals It Is a Phytophthora Species With Downy Mildew-Like Characteristics

On the basis of its downy mildew-like morphology, the litchi downy blight pathogen was previously named Peronophythora litchii. Recently, however, it was proposed to transfer this pathogen to Phytophthora clade 4. To better characterize this unusual oomycete species and important fruit pathogen, we obtained the genome sequence of Phytophthora litchii and compared it to those from other oomycete species. P. litchii has a small genome with tightly spaced genes. On the basis of a multilocus phylogenetic analysis, the placement of P. litchii in the genus Phytophthora is strongly supported. Effector proteins predicted included 245 RxLR, 30 necrosis-and-ethylene-inducing protein-like, and 14 crinkler proteins. The typical motifs, phylogenies, and activities of these effectors were typical for a Phytophthora species. However, like the genome features of the analyzed downy mildews, P. litchii exhibited a streamlined genome with a relatively small number of genes in both core and species-specific protein families. The low GC content and slight codon preferences of P. litchii sequences were similar to those of the analyzed downy mildews and a subset of Phytophthora species. Taken together, these observations suggest that P. litchii is a Phytophthora pathogen that is in the process of acquiring downy mildew-like genomic and morphological features. Thus P. litchii may provide a novel model for investigating morphological development and genomic adaptation in oomycete pathogens.

Development of a sensitive nested-polymerase chain reaction (PCR) assay for the detection of Ustilago scitaminea

A species-specific polymerase chain reaction (PCR) assay was developed for rapid and accurate detection of Ustilago scitaminea, the causal agent of sugarcane smut disease. Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of U. scitaminea, a pair of speciesspecific primers, SL1 (5`-CAGTGCACGAAAGTACCTGTGG-3`) and SR2 (5`CTAGGGCGGTGTTCAGAAGCAC-3`) was designed by using a panel of fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique PCR product about 530 bp in length from U. scitaminea strains with a detecting sensitivity at 200 fg of the fungal genomic DNA in a 25 μl reaction solution. To increase sensitivity, a nested-PCR protocol was further established, which used ITS4/ITS5 as the first-round primers followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by 10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag. The nested-PCR detected U. scitaminea from young sugarcane leaves with no visible smut disease symptoms. The findings from this study provide a sensitive and reliable technique for the early detection of U. scitaminea, which would be useful for sugarcane quarantine and production of germfree seedcanes.

A Puf RNA-binding protein encoding gene PlM90 regulates the sexual and asexual life stages of the litchi downy blight pathogen Peronophythora litchii

Sexual and asexual reproduction are two key processes in the pathogenic cycle of many filamentous pathogens. However in Peronophythora litchii, the causal pathogen for the litchi downy blight disease, critical regulator(s) of sexual or asexual differentiation has not been elucidated. In this study, we cloned a gene named PlM90 from P. litchii, which encodes a putative Puf RNA-binding protein. We found that PlM90 was highly expressed during asexual development, and much higher than that during sexual development, while relatively lower during cyst germination and plant infection. By polyethylene glycol (PEG)-mediated protoplast transformation, we generated three PlM90-silenced transformants and found a severely impaired ability in sexual spore production and a delay in stages of zoospore release and encystment. However, the pathogenicity of P. litchii was not affected by PlM90-silencing. Therefore we conclude that PlM90 specifically regulates the sexual and asexual differentiation of P. litchii.

Antifungal Activity of Natural Volatile Organic Compounds against Litchi Downy Blight Pathogen Peronophythora litchii

Litchi (Litchi chinensis Sonn.) is a commercially important fruit but its production and quality are restricted by litchi downy blight, caused by the oomycete pathogen Peronophythora litchii Chen. Volatile substances produced by a biocontrol antinomycetes Streptomyces fimicarius BWL-H1 could inhibited P. litchii growth and development both in vitro and in detached litchi leaf and fruit infection assay. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) analyses indicated that volatile organic compounds (VOCs) from BWL-H1 resulted in severe damage to the endomembrane system and cell wall of P. litchii cells in vitro and abnormal morphology of appressoria, as well as deformed new hyphae in infection process. VOCs could suppress mycelial growth, sporulation, while with no obvious effect on sporangia germination. Based on gas chromatography-mass spectrophotometric analyses, 32 VOCs were identified from S. fimicarius BWL-H1, the most abundant of which was phenylethyl alcohol. Eight VOCs, including phenylethyl alcohol, ethyl phenylacetate, methyl anthranilate, α-copaene, caryophyllene, humulene, methyl salicylate and 4-ethylphenol, that are commercially available, were purchased and their bioactivity was tested individually. Except for humulene, the other seven tested volatile compounds shown strong inhibitory activity against mycelial growth, sporulation, sporangia germination and germ-tube growth of P. litchii. Especially, 4-ethylphenol showed the highest inhibitory effect on sporulation at a very low concentration of 2 µL/L. Overall, our results provided a better understanding of the mode of action of volatiles from BWL-H1 on P. litchii, and showed that volatiles from BWL-H1 have the potential for control of postharvest litchi downy blight.

Synergistic Effects of Resveratrol and Pyrimethanil against Botrytis cinerea on Grape

Botrytis cinerea is the pathogen of gray mold disease affecting a wide range of plant hosts, with consequential economic losses worldwide. The increased frequency of fungicide resistance of the pathogen challenges its disease management, and thus the development of alternative control strategies are urgently required. In this study, we showed excellent synergistic interactions between resveratrol and pyrimethanil. Significant synergistic values were recorded by the two-drug combination on the suppression of mycelial growth and conidia germination of B. cinerea. The combination of resveratrol and pyrimethanil caused malformation of mycelia. Moreover, the inoculation assay was conducted on table grape and consistent synergistic suppression of the two-drug combination was found in vivo. Our findings first revealed that the combination of resveratrol and pyrimethanil has synergistic effects against resistant B. cinerea and support the potential use of resveratrol as a promising adjuvant on the control of gray mold.