Artificial trans-kingdom RNAi of FolRDR1 is a potential strategy to control tomato wilt disease.

Tomato is cultivated worldwide as a nutrient-rich vegetable crop. Tomato wilt disease caused by Fusarium oxysporum f.sp. Lycopersici (Fol) is one of the most serious fungal diseases posing threats to tomato production. Recently, the development of Spray-Induced Gene Silencing (SIGS) directs a novel plant disease management by generating an efficient and environmental friendly biocontrol agent. Here, we characterized that FolRDR1 (RNA-dependent RNA polymerase 1) mediated the pathogen invasion to the host plant tomato, and played as an essential regulator in pathogen development and pathogenicity. Our fluorescence tracing data further presented that effective uptakes of FolRDR1-dsRNAs were observed in both Fol and tomato tissues. Subsequently, exogenous application of FolRDR1-dsRNAs on pre-Fol-infected tomato leaves resulted in significant alleviation of tomato wilt disease symptoms. Particularly, FolRDR1-RNAi was highly specific without sequence off-target in related plants. Our results of pathogen gene-targeting RNAi have provided a new strategy for tomato wilt disease management by developing an environmentally-friendly biocontrol agent.

Suppression of Rice Osa-miR444.2 Improves the Resistance to Sheath Blight in Rice Mediating through the Phytohormone Pathway.

MicroRNAs (miRNAs) are a class of conserved small RNA with a length of 21-24 nucleotides in eukaryotes, which are involved in development and defense responses against biotic and abiotic stresses. By RNA-seq, Osa-miR444b.2 was identified to be induced after Rhizoctonia solani (R. solani) infection. In order to clarify the function of Osa-miR444b.2 responding to R. solani infection in rice, transgenic lines over-expressing and knocking out Osa-miR444b.2 were generated in the background of susceptible cultivar Xu3 and resistant cultivar YSBR1, respectively. Over-expressing Osa-miR444b.2 resulted in compromised resistance to R. solani. In contrast, the knocking out Osa-miR444b.2 lines exhibited improved resistance to R. solani. Furthermore, knocking out Osa-miR444b.2 resulted in increased height, tillers, smaller panicle, and decreased 1000-grain weight and primary branches. However, the transgenic lines over-expressing Osa-miR444b.2 showed decreased primary branches and tillers, but increased panicle length. These results indicated that Osa-miR444b.2 was also involved in regulating the agronomic traits in rice. The RNA-seq assay revealed that Osa-miR444b.2 mainly regulated the resistance to rice sheath blight disease by affecting the expression of plant hormone signaling pathways-related genes such as ET and IAA, and transcription factors such as WRKYs and F-boxes. Together, our results suggest that Osa-miR444b.2 negatively mediated the resistance to R. solani in rice, which will contribute to the cultivation of sheath blight resistant varieties.

Fol-milR1, a pathogenicity factor of Fusarium oxysporum, confers tomato wilt disease resistance by impairing host immune responses.

Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection. Here, using sRNA-seq, we demonstrate that Fol-milR1, a trans-kingdom small RNA, is exported into tomato cells after infection. The knockout strain ∆Fol-milR1 displays attenuated pathogenicity to the susceptible tomato cultivar 'Moneymaker'. On the other hand, Fol-milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar 'Motelle'. Several tomato mRNAs are predicted targets of Fol-milR1. Among these genes, Solyc06g007430 (encoding the CBL-interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol-milR1. Furthermore, SlyFRG4 loss-of-function alleles created using CRISPR/Cas9 in tomato ('Motelle') exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol-milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus-induced gene silenced (VIGS) knock-down SlyAGO4a plants exhibit reduced susceptibility to Fol. Together, our findings support a model in which Fol-milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection.

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30 nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 µg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

FolVps9, a Guanine Nucleotide Exchange Factor for FolVps21, Is Essential for Fungal Development and Pathogenicity in Fusarium oxysporum f. sp. lycopersici.

The soil-borne, asexual fungus Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of tomato wilt disease. Autophagy plays a crucial role in the development and virulence of Fol. The Fol endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity. Rab GTPases and the regulators are highly conserved in regulating autophagy and endocytosis in most eukaryotes. Identification and characterization of additional Rab regulators in fungal pathogens should facilitate the understanding of the autophagy and endocytosis in different filamentous fungi. Here, we have identified and characterized the yeast VPS9 homolog FolVPS9 in Fol. Targeted gene deletion showed that FolVPS9 is important for growth, conidiation and virulence in Fol. Cytological examination revealed that FolVps9 co-localized with FolVps21 (a marker of early endosome) and played a critical role in endocytosis and autophagosome degradation. Pull-down assays showed that FolVps9 interacted with FolVps21, which was also important for development and plant infection in Fol. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays revealed that FolVps9 specifically interacts with the GDP-bound form of FolVps21. Furthermore, a constitutively active form of FolVps21 (Q72L) was able to rescue defects of ΔFolvps9 and ΔFolvps21 mutants. In summary, our study provides solid evidence that FolVps9 acts as a FolVps21 guanine nucleotide exchange factor (GEFs) to modulate endocytosis and autophagy, thereby controlling vegetative growth, asexual development and pathogenicity in Fol.

The SNARE protein FolVam7 mediates intracellular trafficking to regulate conidiogenesis and pathogenicity in Fusarium oxysporum f. sp. lycopersici.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are conserved in fungi, plants and animals. The Vam7 gene encodes a v-SNARE protein that involved in vesicle trafficking in fungi. Here, we identified and characterized the function of FolVam7, a homologue of the yeast SNARE protein Vam7p in Fusarium oxysporum f. sp. lycopersici (Fol), a fungal pathogen of tomato. FolVam7 contains SNARE and PX (Phox homology) domains that are indispensable for normal localization and function of FolVam7. Targeted gene deletion showed that FolVam7-mediated vesicle trafficking is important for vegetative growth, asexual development, conidial morphology and plant infection. Further cytological examinations revealed that FolVam7 is localized to vesicles and vacuole membranes in the hyphae stage. Moreover, the ΔFolvam7 mutant is insensitive to salt and osmotic stresses and hypersensitive to cell wall stressors. Taken together, our results suggested that FolVam7-mediated vesicle trafficking promotes vegetative growth, conidiogenesis and pathogenicity of Fol.

An integrated analysis of mRNA and sRNA transcriptional profiles in tomato root: Insights on tomato wilt disease.

Tomato wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL) is a worldwide destructive disease of tomato. As exploring gene expression and function approaches constitute an initial point for investigating pathogen-host interaction, we performed RNA-seq and sRNA-seq analysis to investigate the transcriptome of tomato root under FOL infection. Differentially expressed (DE) protein-coding gene and miRNA gene profiles upon inoculation with FOL were presented at twenty-four hours post-inoculation in four treatments. A total of more than 182.6 million and 132.2 million high quality clean reads were obtained by RNA-seq and sRNA-seq, respectively. A large overlap was found in DE mRNAs between susceptible cultivar Moneymaker and resistant cultivar Motelle. Gene Ontology terms were mainly classified into catalytic activity, metabolic process and binding. Combining with qRT-PCR and Northern blot, we validated the transcriptional profile of five genes and five miRNAs conferred to FOL infection. Our work allowed comprehensive understanding of different transcriptional reaction of genes/miRNAs between the susceptible and resistant cultivars tomato to the FOL challenge, which could offer us with a future direction to generate models of mediated resistance responses.

FRG3, a Target of slmiR482e-3p, Provides Resistance against the Fungal Pathogen Fusarium oxysporum in Tomato.

The vast majority of plant disease resistance (R) genes encode nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins, which specifically determine the plant immune response and have been demonstrated to be targets of several microRNA (miRNA) families. The fungus Fusarium oxysporum f. sp. lycopersici (FOL) causes vascular wilt disease in tomato worldwide. Here, we explored a possible role for FGR3 in tomato defense against FOL. FRG3 is a predicted NBS-LRR like gene that is targeted by slmiR482e-3p, a member of slmiR482 miRNA family. Northern blot data demonstrated that all seven members of the slmiR482 family were regulated in diverse ways after infection by FOL. The ability of FRG3 to be regulated by slmiR482e-3p was confirmed at the transcript level by co-expression studies in Nicotiana benthamiana. A virus-induced gene silencing (VIGS) approach revealed that FRG3 confers resistance to the Motelle tomato cultivar. Taken together, our study has identified a novel R gene, FRG3, which is targeted by slmiR482e-3p at the transcript level, and is necessary for resistance to tomato wilt disease in planta.

T-type calcium channel antagonists, mibefradil and NNC-55-0396 inhibit cell proliferation and induce cell apoptosis in leukemia cell lines.

BACKGROUND: T-type Ca(2+) channels are often aberrantly expressed in different human cancers and participate in the regulation of cell cycle progression, proliferation and death. METHODS: RT-PCR, Q-PCR, western blotting and whole-cell patch-clamp recording were employed to assess the expression of T-type Ca(2+) channels in leukemia cell lines. The function of T-type Ca(2+) channels in leukemia cell growth and the possible mechanism of the effect of T-type Ca(2+) channel antagonists on cell proliferation and apoptosis were examined in T-lymphoma cell lines. RESULTS: We show that leukemia cell lines exhibited reduced cell growth when treated with T-type Ca(2+) channel inhibitors, mibefradil and NNC-55-0396 in a concentration-dependent manner. Mechanistically, these inhibitors played a dual role on cell viability: (i) blunting proliferation, through a halt in the progression to the G1-S phase; and (ii) promoting cell apoptosis, partially dependent on the endoplasmic reticulum Ca(2+) release. In addition, we observed a reduced phosphorylation of ERK1/2 in MOLT-4 cells in response to mibefradil and NNC-55-0396 treatment. CONCLUSIONS: These results indicate that mibefradil and NNC-55-0396 regulate proliferation and apoptosis in T-type Ca(2+) channel expressing leukemia cell lines and suggest a potential therapeutic target for leukemia.

Identification and functional characterization of voltage-gated sodium channels in lymphocytes.

A variety of ion channels has been discovered in lymphocytes. RT-PCR and real-time PCR analysis revealed that ALL (acute lymphocytic leukemia) cell lines and human peripheral blood mononuclear cells mainly expressed TTX (tetrodotoxin)-sensitive voltage-gated sodium channels (VGSCs). Expression of VGSC protein was confirmed by western blots and Immunofluorescence. Whole-cell patch-clamp recordings showed that a sub-population (20%) of MOLT-4 cells expressed functional VGSCs, which were TTX-sensitive. Importantly, 2 µM TTX decreased the invasion of Jurkat and MOLT-4 cells ∼90%. These results indicate that the activity of VGSCs could represent a novel mechanism potentiating the invasive capacity of these cells.

Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants.

Receptor-like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine-rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn(2+), and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter-GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H(2)O(2) treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock-out mutants sik1-1 and sik1-2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1-overexpressing plants. Also, the accumulation of H(2)O(2) in leaves of OsSIK1-overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3'-diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system.

Effect of quercetin on colon contractility and L-type Ca(2+) channels in colon smooth muscle of guinea-pig.

The aim of the present study was to investigate the effects of quercetin on colon contractility and voltage-dependent Ca(2+) channels in the single smooth muscle cell isolated from the proximal colon of guinea-pig and to clarify whether its effect on L-type Ca(2+) current (I(Ca,L)) would be related to its myorelaxing properties. Colon smooth muscle strips were used to take contractile tension recordings. Smooth muscle cells were freshly isolated from the proximal colon of guinea-pig by means of papain treatment. I(Ba,L) (barium instead of calcium as current carrier) was measured by using whole-cell patch-clamp techniques. The results showed that quercetin relaxed colon muscle strips in a concentration-dependent manner and antagonized the contractile effect of acetylcholine and neostigmine. Preincubation with indomethcin [cyclooxygenase (COX) inhibitor] and methylene blue [guanylate cyclase (GC) inhibitor] significantly attenuated the relaxing effect of quercetin, respectively. Quercetin increased I(Ba,L) in a concentration- [EC(50)= (7.59+/-0.38) mumol/L] and voltage-dependent pattern, and shifted the maximum of the current-voltage curve by 10 mV in the depolarizing direction without modifying the threshold potential for Ca(2+) influx. Quercetin shifted the steady-state inactivation curve toward more positive potentials by approximately 3.75 mV without affecting the slope of activation and inactivation curve. H-89 (PKA inhibitor) abolished quercetin-induced I(Ba,L) increase, while cAMP enhanced the quercetin-induced I(Ba,L) increase. The patch-clamp results proved that quercetin increased I(Ba,L) via PKA pathway. It is therefore suggested that the relaxing effect of quercetin attributes to the interaction of GC and COX stimulation, as well as the antagonism effect on acetylcholine, which hierarchically prevails over the increase in the Ca(2+) influx to be expected from I(Ca,L) stimulation.

[The characteristics and oxidative modulation of large-conductance calcium-activated potassium channels in guinea-pig colon smooth muscle cells.].

To investigate the characteristics of large-conductance calcium-activated potassium channels (BK(Ca)) and the effect of hydrogen peroxide (H2O2) on BK(Ca) in guinea-pig proximal colon smooth muscle cells, single smooth muscle cells of guinea-pig colon were enzymatically isolated in low calcium solution containing papain (3 mg/mL), DTT (2 mg/mL), and bovine serum albumin (BSA, 2 mg/mL). Tissues were incubated at 36 degrees C in enzyme solution for 15 min and were then suspended in enzyme-free low calcium solution. Inside-out single channel recording technique was used to record BK(Ca) current. The intracellular (bath) and microelectrode solution both contained symmetrical high potassium. The BK(Ca) in guinea-pig colon smooth muscle cell possesses: 1) voltage-dependence, 2) high selectivity for potassium ion, 3) large conductance (223.7 pS+/-9.2 pS), 4) dependence of [Ca(2+)](i). Intracellular application of H2O2 decreased the open probability (P(o)) of BK(Ca) at low concentration (

Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.

AIM: To investigate the action of genistein (GST), a broad spectrum tyrosine kinase inhibitor, on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells. METHODS: Smooth muscle cells in guinea pig proximal colon were enzymatically isolated. Nystatin-perforated whole cell patch clamp technique was used to record potassium currents including fast transient outward current (IKto) and delayed rectifier current (IKdr), two of which were isolated pharmacologically with 10 mmol/L tetraethylammonium or 5 mmol/L 4-aminopyridine. Contamination of calcium-dependent potassium currents was minimized with no calcium and 0.2 mmol/L CdCl2 in an external solution. RESULTS: GST (10-100 micromol/L) reversibly and dose-dependently reduced the peak amplitude of IKto with an IC50 value of 22.0+/-6.9 micromol/L. To a lesser extent, IKdr was also inhibited in both peak current and sustained current. GST could not totally block the outward potassium current as a fraction of the outward potassium current, which was insensitive to GST. GST had no effect on the steady-state activation (n=6) and inactivation kinetics (n=6) of IKto. Sodium orthovanadate (1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited GST-induced inhibition (P<0.05). CONCLUSION: GST can dose-dependently and reversibly block voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.