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The soil-borne ascomycete fungus Verticillium dahliae causes vascular wilt disease and can seriously diminish the yield and quality of important crops. Functional analysis of growth- and pathogenicity-related genes is essential for revealing the pathogenic molecular mechanism of V. dahliae. Phospholipase is an important virulence factor in fungi that hydrolyzes phospholipids into fatty acid and other lipophilic substances and is involved in hyphal development. Thus far, only a few V. dahliae phospholipases have been identified, and their involvement in V. dahliae development and pathogenicity remains unknown. In this study, the function of the patatin-like phospholipase gene in V. dahliae (VdPLP, VDAG_00942) is characterized by generating gene knockout and complementary mutants. Vegetative growth and conidiation of VdPLP deletion mutants (ΔVdPLP) were significantly reduced compared with wild type and complementary strains, but more microsclerotia formed. The ΔVdPLP mutants were very sensitive to the cell-wall-perturbing agents: calcofluor white (CFW) and Congo red (CR). The transcriptional level of genes related to the cell wall integrity (CWI) pathway and chitin synthesis were downregulated, suggesting that VdPLP has a pivotal role in the CWI pathway and chitin synthesis in V. dahliae. ΔVdPLP strains were distinctly impaired in in their virulence and ability to colonize Nicotiana benthamiana roots. Our results demonstrate that VdPLP regulates hyphal growth and conidial production and is involved in stabilizing the cell wall, thus mediating the pathogenicity of V. dahliae.

Sweet cherry (Prunus avium L.) is an important fruit crop in which fruit size is strongly associated with commercial value; few genes associated with fruit size have, however, been identified in sweet cherry. Members of the CYP78A subfamily, a group of important cytochrome P450s, have been found to be involved in controlling seed size and development in Arabidopsis thaliana, rice, soybean, and tomato. However, the influence of CYP78A members in controlling organ size and the underlying molecular mechanisms in sweet cherry and other fruit trees remains unclear. Here, we characterized a P. avium CYP78A gene PaCYP78A9 that is thought to be involved in the regulation of fruit size and organ development using overexpression and silencing approaches. PaCYP78A9 was significantly expressed in the flowers and fruit of sweet cherry. RNAi silencing of PaCYP78A9 produced small cherry fruits and PaCYP78A9 was found to affect fruit size by mediating mesocarp cell proliferation and expansion during fruit growth and development. Overexpression of PaCYP78A9 in Arabidopsis resulted in increased silique and seed size and PaCYP78A9 was found to be highly expressed in the inflorescences and siliques of transgenic plants. Genes related to cell cycling and proliferation were downregulated in fruit from sweet cherry TRV::PaCYP78A9-silencing lines, suggesting that PaCYP78A9 is likely to be an important upstream regulator of cell cycle processes. Together, our findings indicate that PaCYP78A9 plays an essential role in the regulation of cherry fruit size and provide insights into the molecular basis of the mechanisms regulating traits such as fruit size in P. avium.

Sweet cherry (Prunus avium L.) is an economically important fruit crop, and has a variety of health promoting benefits. Because of sweet cherry tree possessing strong apical dominance, shoot branching, as a significant agriculture trait, determines fruit tree architecture and occupies an important position in sweet cherry yield. However, the mechanism that regulated sweet cherry shoot branching remains largely unknown. Here, we isolated and functionally characterized a sweet cherry gene homologous to the rice GNS4 gene, named PaCYP724B1. Spatial-temporal expression analysis revealed that PaCYP724B1 was expressed with a particularly high expression in the axillary shoots tissues during the shoot branching formation. Furthermore, overexpression of PaCYP724B1 in transgenic Arabidopsis plants enhanced shoot branching. In contrast, Silencing of NbCYP724B1 in Nicotiana tabacum via virus-induced gene silencing technology with heterologous PaCYP724B1 gene fragment resulted in a significantly inhibited shoot branching phenotype. Subcellular localization assays demonstrated that PaCYP724B1 protein was localized on the endoplasmic reticulum (ER). Taken together, the results denote new insights into the important role of PaCYP724B1 regulating sweet cherry shoot branching.

Background Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. Results High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs—19-nt duplex with 2-nt 3′ overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)—into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. Conclusion Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0287-4) contains supplementary material, which is available to authorized users.

Verticillium dahliae causes a serious wilt disease of important crops and is difficult to control. Few plasma-membrane transport proteins for nutrient acquisition have been identified for this fungus, and their involvement in the disease process is unknown. Here, a plasma-membrane protein, the V. dahliae thiamine transporter protein VdThit, was characterized functionally by deletion of the VdThit gene in V. dahliae. Disruption strains were viable, but growth and conidial germination and production were reduced and virulence was impaired. Interestingly, by supplementing exogenous thiamine, growth, conidiation, and virulence of the VdΔThit mutants were partially restored. Stress-tolerance assays showed that the VdΔThit mutant strains were markedly more susceptible to oxidative stress and UV damage. High-pressure liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) analyses showed low levels of pyruvate metabolism intermediates acetoin and acetyl coenzyme A (acetyl-CoA) in the VdΔThit mutant strains, suggesting that pyruvate metabolism was suppressed. Expression analysis of VdThit confirmed the importance of VdThit in vegetative growth, reproduction, and invasive hyphal growth. Furthermore, a green fluorescent protein (GFP)-labeled VdΔThit mutant (VdΔThit-7-GFP) was suppressed in initial infection and root colonization, as viewed with light microscopy. Together, these results showed that VdThit plays an indispensable role in the pathogenicity of V. dahliae.