Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine γ-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars.

The use of transgenic plants in agriculture provides many economic benefits, but it also raises concerns over the potential impact of transgenic plants on the environment. We here examined the impact of transgenic high-methionine soybean ZD91 on the arbuscular mycorrhizal (AM) fungal community structure in rhizosphere soil. Our investigations based on clone libraries were conducted in field trials at four growth stages of the crops each year from 2012 to 2013. A total of 155 operational taxonomic units (OTUs) of AM fungi were identified based on the sequences of small subunit ribosomal RNA (SSU rRNA) genes. There were no significant differences found in AM fungal diversity in rhizosphere soil during the same growth stage between transgenic soybean ZD91 and its non-transgenic parental soybean ZD. In addition, plant growth stage and year had the strongest effect on the AM fungal community structure while the genetically modified (GM) trait studied was the least explanatory factor. In conclusion, we found no indication that transgenic soybean ZD91 cultivation poses a risk for AM fungal communities in agricultural soils.

Hexokinases are conserved proteins functioning in glucose sensing and signaling. The rice blast fungus Magnaporthe oryzae contains several hexokinases, including MoHxk1 (hexokinase) and MoGlk1 (glucokinase) encoded respectively by MoHXK1 and MoGLK1 genes. The heterologous expression of MoGlk1 and MoHxk1 in Saccharomyces cerevisiae confirmed their conserved functions. Disruption of MoHXK1 resulted in growth reduction in medium containing fructose as the sole carbon source, whereas disruption of MoGLK1 did not cause the similar defect. However, the ΔMoglk1 mutant displayed decreased proton extrusion and a lower biomass in the presence of ammonium, suggesting a decline in the utilization of ammonium. Additionally, the MoGLK1 allele lacking catalytic activity restored growth to the ΔMoglk1 mutant. Moreover, the expression of MoPMA1 encoding a plasma membrane H(+)-ATPase decreased in the ΔMoglk1 mutant that can be suppressed by glucose and G-6-P. Thus, MoGlk1, but not MoHxk1, regulates ammonium utilization through a mechanism that is independent from its catalytic activity.

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular vesicle fusion, which is an essential cellular process of the eukaryotic cells. To investigate the role of SNARE proteins in the rice blast fungus Magnaporthe oryzae, MoSec22, an ortholog of Saccharomyces cerevisiae SNARE protein Sec22, was identified and the MoSEC22 gene disrupted. MoSec22 restored a S. cerevisiae sec22 mutant in resistance to cell wall perturbing agents, and the ΔMosec22 mutant also exhibited defects in mycelial growth, conidial production, and infection of the host plant. Treatment with oxidative stress inducers indicated a breach in cell wall integrity, and staining and quantification assays suggested abnormal chitin deposition on the lateral walls of hyphae of the ΔMosec22 mutant. Furthermore, hypersensitivity to the oxidative stress correlates with the reduced expression of the extracellular enzymes peroxidases and laccases. Our study thus provides new evidence on the conserved function of Sec22 among fungal organisms and indicates that MoSec22 has a role in maintaining cell wall integrity affecting the growth, morphogenesis, and virulence of M. oryzae.

Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.