Verticillium wilt caused by the soil-borne fungus Verticillium dahliae is a common, devastating plant vascular disease notorious for causing economic losses. Despite considerable research on plant resistance genes, there has been little progress in modeling the effects of this fungus owing to its complicated pathogenesis. Here, we analyzed the transcriptional and metabolic responses of Arabidopsis thaliana to V. dahliae inoculation by Illumina-based RNA sequencing (RNA-seq) and nuclear magnetic resonance (NMR) spectroscopy. We identified 13,916 differentially expressed genes (DEGs) in infected compared with mock-treated plants. Gene ontology analysis yielded 11,055 annotated DEGs, including 2,308 for response to stress and 2,234 for response to abiotic or biotic stimulus. Pathway classification revealed involvement of the metabolic, biosynthesis of secondary metabolites, plant–pathogen interaction, and plant hormone signal transduction pathways. In addition, 401 transcription factors, mainly in the MYB, bHLH, AP2-EREBP, NAC, and WRKY families, were up- or downregulated. NMR analysis found decreased tyrosine, asparagine, glutamate, glutamine, and arginine and increased alanine and threonine levels following inoculation, along with a significant increase in the glucosinolate sinigrin and a decrease in the flavonoid quercetin glycoside. Our data reveal corresponding changes in the global transcriptomic and metabolic profiles that provide insights into the complex gene-regulatory networks mediating the plant’s response to V. dahliae infection.

BackgroundLarge 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.ResultsHigh-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.ConclusionOur quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0287-4) contains supplementary material, which is available to authorized users.

BACKGROUD Volatile terpenes can act as ecological signals to affect insect behavior. It has been proposed that the manipulation of terpenes in plants can help to control herbivore pests. In order to investigate the potential pest management function of (E)‐β‐caryophyllene in cotton plants, the (E)‐β‐caryophyllene synthase gene (GhTPS1) was inserted into Gossypium hirsutum variety R15 to generate overexpression lines. RESULTS Four GhTPS1‐transgenic lines were generated, and GhTPS1 expression in transgenic L18 and L46 lines was 3‐5‐fold higher than in R15 plants. The transgenic L18 and L46 lines also emitted significantly more (E)‐β‐caryophyllene than R15. In laboratory bioassays, L18 and L46 plants reduced pests Apolygus lucorum, Aphis gossypii and Helicoverpa armigera, and attracted parasitoids Peristenus spretus and Aphidius gifuensis, but not Microplitis mediator. In open‐field trials, L18 and L46 plants reduced A. lucorum, Adelphocoris suturalis and H. armigera, but had no significant effects on predators. CONCLUSION Our findings suggest that L18 and L46 plants reduce several major hemipteran and lepidopteran cotton pests, whereas, two parasitoids P. spretus and A. gifuensis, were attracted by L18 and L46 plants. This study shows that overexpressing GhTPS1 in cotton may help to improve pest management in cotton fields. © 2019 Society of Chemical Industry

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.

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Rhizosphere microorganisms are closely associated with phosphorus (P) uptake in plants and are considered potential agents to mitigate P shortage. However, the mechanisms of rhizospheric microbial community assembly under P deficiency have yet to be elucidated. In this study, bacterial and fungal communities in rice rhizosphere and their P mobilization potential under high (+P) and low (−P) concentrations of P were investigated. Bacterial and fungal community structures were significantly different between −P and +P treatments. And both bacterial and fungal P-mobilizing taxa were enriched in-P treatment; however, the proportion of P-mobilizing agents in the fungal community was markedly greater than that in the bacterial community. A culture experiment confirmed that microbial phosphate solubilizing capacity was significantly higher in −P treatment compared with that in +P treatment. −P treatment lowered bacterial diversity in rice rhizosphere but increased fungal diversity. Further analysis demonstrated that the contribution of deterministic processes in governing bacterial community assembly was strengthened under P deficiency but was largely weakened in shaping the fungal community. These results highlighted that enriching P-mobilizing microbes in the rhizosphere is a vital way for rice to cope with P deficiency, and that fungi contribute considerably to P mobilization in rice rhizosphere. Findings from the study provide novel insights into the assembly of the rhizosphere microbiome under P deficiency and this will facilitate the development of rhizosphere microbial regulation strategies to increase nutrient uptake in plants.

Verticillium wilt, caused by the ascomycete fungus Verticillium dahliae (Vd), is a devastating disease of numerous plant species. However, the pathogenicity/virulence-related genes in this fungus, which may be potential targets for improving plant resistance, remain poorly elucidated. For the study of these genes in Vd, we used a well-established host-induced gene silencing (HIGS) approach and identified 16 candidate genes, including a putative adenylate kinase gene (VdAK). Transiently VdAK-silenced plants developed milder wilt symptoms than control plants did. VdAK-knockout mutants were more sensitive to abiotic stresses and had reduced germination and virulence on host plants. Transgenic Nicotiana benthamiana and Arabidopsis thaliana plants that overexpressed VdAK dsRNAs had improved Vd resistance than the wild-type. RT-qPCR results showed that VdAK was also crucial for energy metabolism. Importantly, in an analysis of total small RNAs from Vd strains isolated from the transgenic plants, a small interfering RNA (siRNA) targeting VdAK was identified in transgenic N. benthamiana. Our results demonstrate that HIGS is a promising strategy for efficiently screening pathogenicity/virulence-related genes of Vd and that VdAK is a potential target to control this fungus.

The unscientific application of pesticides can easily cause a series of ecological environmental safety issues, which seriously restrict the sustainable development of modern agriculture. The great progress in nanotechnology has allowed the continuous development of plant protection strategies. The nanonization and delivery of pesticides offer many advantages, including their greater absorption and conduction by plants, improved efficacy, reduced dosage, delayed resistance, reduced residues, and protection from natural enemies and beneficial insects. In this review, we focus on the recent advances in multifunctional nanoparticles and nanopesticides. The definition of nanopesticides, the types of nanoparticles used in agriculture and their specific synergistic mechanisms are introduced, their safety is evaluated, and their future application prospects, about which the public is concerned, are examined.