FreB is involved in the ferric metabolism and multiple pathogenicity-related traits of Verticillium dahliae.

Ferric reductases are integral membrane proteins involved in the reduction of environmental ferric iron into the biologically available ferrous iron. In the most overwhelming phytopathogenic fungus, Verticillium dahliae, these ferric reductase are not studied in details. In this study we explored the role of FreB gene (VDAG_06616) in the ferric reduction and virulence of V. dahliae by generating the knockout mutants (ΔFreB) and complementary strains (ΔFreB-C) using protoplast transformation. When cultured on media supplemented with FeSO4, FeCl3 and no iron, ΔFreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of ΔFreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in ΔFreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in ΔFreB. Compared with the Vd-wt and ΔFreB-C, ΔFreB-1 and ΔFreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). ΔFreB-1 and ΔFreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 µM H2O2 was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, ΔFreB exhibited less disease symptoms than Vd-wt and ΔFreB-C. In conclusion, the present findings not only indicate that FreB mediates the ferric metabolism and is required for the full virulence in V. dahliae, but would also accelerate future investigation to uncover the pathogenic mechanism of this fungus.

VdThit, a Thiamine Transport Protein, Is Required for Pathogenicity of the Vascular Pathogen Verticillium dahliae.

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.

[IR and Raman spectra studies of Rotundine based on DFT].

Infrared spectroscopy (IR), the normal Raman spectroscopy (NRS) and the surface enhanced Raman spectroscopy (SERS) in new Ag/Cu nanomaterial of Rotundine were studied in the present paper. The IR and the NRS of Rotundine were calculated by the density functional theory (DFT) using B3LYP/6-311+G(d, p), then the spectral intensity graph of Rotundine were given. The vibrational peaks were assigned comprehensively by the visualization software of Gauss view 5. 0. Rotundine has obvious infrared and Raman vibrational peak in the wave number range of 3 300-2500 and 1 800-600 cm(-1). SnCl2 and PVP was used as capping agent for the silver nanoparticles in SERS of Rotundine. Finally, by using the method of cyclic immersion well dispersed silver nanoparticles was obtained and achieved good enhancement effect. This molecule acquired strong selective enhancement vibration peak, In the wave number ranges of 1 500-1 400 and 1 000-700 cm(-1) the enhancement effect is most obvious. After analyzed, the methylene of this molecule is adsorbed on the silver nanoparticles surface and the angle between the benzene ring and the silver substrate is close to 90 degrees. The theoretically calculated spectra of Rotundine are consistent with the obtained experimental spectra. There are some differences may be due to the interaction forces between molecules and so on. The visualization software displayed the structure characteristics and molecular group vibration of this molecular visually and provided important basis for assigning the vibrational peaks. Rotundine is an important traditional Chinese medicine agent contained in many kinds of sedative drugs. The study provides a strong basis for the rapid, feature and trace identification of Rotundine and also supplies important reference for the biological role of central inhibition of analgesic drugs.

Comparative analyses reveal potential uses of Brachypodium distachyon as a model for cold stress responses in temperate grasses.

BACKGROUND: Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS: Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS: We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.

The low temperature-responsive, Solanum CBF1 genes maintain high identity in their upstream regions in a genomic environment undergoing gene duplications, deletions, and rearrangements.

Some plants like Arabidopsis thaliana increase in freezing tolerance when exposed to low nonfreezing temperatures, a process known as cold acclimation. Other plants including tomato, Solanum lycopersicum, are chilling sensitive and incur injury during prolonged low temperature exposure. A key initial event that occurs upon low temperature exposure is the induction of genes encoding the CBF transcription factors. In Arabidopsis three CBF genes, present in a tandemly-linked cluster, are induced by low temperatures. Tomato also harbors three tandemly-linked CBF genes, Sl-CBF3-CBF1-CBF2, but only one of these, Sl-CBF1, is low-temperature responsive. Here we report that Solanum species that are closely-allied to cultivated tomato essentially share this structural organization, but the locus is in a dynamic state of flux. Additional paralogs and in-frame deletions between adjacent genes occur, and the genomic regions flanking the CBF genes are dissimilar across Solanum species. Nevertheless, the CBF1 upstream region remains intact and highly conserved. This feature differed for CBF2 and CBF3, whose upstream regions were far less conserved. CBF1 was also the only low-temperature responsive gene in the cluster and its expression was greatly affected by a circadian clock. The tuber-bearing S. tuberosum and S. commersonii also harbored a fourth gene, CBF4, which was also low temperature responsive. CBF4 was physically linked to CBF5 in S. tuberosum, but CBF5 was absent from S. commersonii. Phylogenic analyses suggest that CBF5-CBF4 resulted from the duplication of the CBF3-CBF1-CBF2 cluster. DNA sequence motifs shared between the Solanum CBF1 and CBF4 upstream regions were identified, portions of which were also present in the Arabidopsis CBF1-3 upstream regions. These results suggest that much greater functional constraints are placed upon the Solanum CBF1 upstream regions over the other CBF upstream regions and that CBF4 has retained the capacity for low temperature responsiveness following the duplication event that gave rise to CBF4.