Transporter-encoding Genes Research Articles

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Overlapping coactivator function is required for transcriptional activation by the Candida glabrata Pdr1 transcription factor.

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and 2 different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and 2 different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high-level Pdr1-dependent gene transcription.

Overlapping coactivator function is required for transcriptional activation by the Candida glabrata Pdr1 transcription factor.

Azole resistance in the pathogenic yeast Candida glabrata is a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding gene CDR1 . Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactive PDR1 alleles. While the identity of hyperactive PDR1 alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification (TAP)-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and two different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and two different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking either SNF2 or SPT7 exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect in PDR1 autoregulation, revealing that these complexes work together to ensure high level Pdr1-dependent gene transcription.

Loss of a conserved C-terminal region of the Aspergillus fumigatus AtrR transcriptional regulator leads to a gene-specific defect in target gene expression.

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologues consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA-seq, we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced.

In Saccharomyces cerevisiae ρ0 Cells, UME6 Contributes to the Activation of ABC Transporter Genes and Pleiotropic Drug Resistance via RPD3 and PDR3

In Saccharomyces cerevisiae, the Rpd3L complex includes the histone deacetylase Rpd3 and the DNA binding proteins Ume6 and Ash1 and serves as a transcriptional silencer or enhancer. In S. cerevisiae, the transcription of PDR5, which encodes a major drug efflux pump, and pleiotropic drug resistance (PDR) are hyperactivated by the transcription factor Pdr3 in ρ0/− cells, which lack mitochondrial DNA. We previously showed that RPD3 and UME6 are required for the activation of PDR5 transcription and PDR in S. cerevisiae ρ0 cells. Here, using real-time PCR analysis, we revealed that RPD3 and UME6 are responsible for the activated basal expression of the ABC transporter-encoding genes SNQ2, PDR15, and PDR5 in S. cerevisiae ρ0 cells. Furthermore, using real-time PCR analysis and a spot dilution assay, we found that Ume6 increases the basal expression of PDR5 and PDR15 and induces PDR in a manner dependent on RPD3 and PDR3 in ρ0 cells. This finding may contribute to the elucidation of the relationships between the molecules required for the activation of ABC transporter genes in S. cerevisiae ρ0/− cells and in pathogenic Candida species.

Transcriptome Analysis of mfs2-Defective Penicillium digitatum Mutant to Reveal Importance of Pdmfs2 in Developing Fungal Prochloraz Resistance.

Demethylation inhibitors (DMIs), including prochloraz, are popular fungicides to control citrus postharvest pathogens such as Penicillium digitatum (green mold). However, many P. digitatum strains have developed prochloraz resistance, which decreases drug efficacy. Specific major facilitator superfamily (MFS) transporter gene mfs2, encoding drug-efflux pump protein MFS2, has been identified in P. digitatum strain F6 (PdF6) to confer fungal strain prochloraz resistance. However, except for the drug-efflux pump function of MFS2, other mechanisms relating to the Pdmfs2 are not fully clear. The present study reported a transcriptome investigation on the mfs2-defective P. digitatum strain. Comparing to the wild-type strain, the mfs2-defective strain showed 717 differentially expressed genes (DEGs) without prochloraz induction, and 1221 DEGs with prochloraz induction. The obtained DEGs included multiple isoforms of MFS transporter-encoding genes, ATP-binding cassette (ABC) transporter-encoding genes, and multidrug and toxic compound extrusion (MATE) family protein-encoding genes. Many of these putative drug-efflux pump protein-encoding genes had significantly lower transcript abundances in the mfs2-defective P. digitatum strain at prochloraz induction, as compared to the wild-type strain, including twenty-two MFS transporter-encoding genes (MFS1 to MFS22), two ABC transporter-encoding genes (ABC1 and ABC2), and three MATE protein-encoding genes (MATE1 to MATE3). The prochloraz induction on special drug-efflux pump protein genes in the wild-type strain was not observed in the mfs2-defective strain, including MFS21, MFS22, ABC2, MATE1, MATE2, and MATE3. On the other hand, the up-regulation of other drug-efflux pump protein genes in the mfs2-defective strain cannot recover the fungal prochloraz resistance, including MFS23, MFS26, MFS27, MFS31, MFS33, and ABC3 to ABC8. The functional enrichment of DEGs based on Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and euKaryotic Orthologous Groups (KOG) database resources suggested some essential contributors to the mfs2-relating prochloraz resistance, including ribosome biosynthesis-related genes, oxidative phosphorylation genes, steroid biosynthesis-related genes, fatty acid and lipid metabolism-related genes, and carbon- and nitrogen-metabolism-related genes. The results indicated that the MFS2 transporter might be involved in the regulation of multiple drug-efflux pump protein gene expressions and multiple metabolism-related gene expressions, thus playing an important role in developing P. digitatum prochloraz resistance.

Far-red light modulates grapevine growth by increasing leaf photosynthesis efficiency and triggering organ-specific transcriptome remodelling

BackgroundGrowing evidence demonstrates that the synergistic interaction of far-red light with shorter wavelength lights could evidently improve the photosynthesis efficiency of multiple species. However, whether/how far-red light affects sink organs and consequently modulates the source‒sink relationships are largely unknown.ResultsHere, equal intensities of white and far-red lights were added to natural light for grape plantlets to investigate the effects of far-red light supplementation on grapevine growth and carbon assimilate allocation, as well as to reveal the underlying mechanisms, through physiological and transcriptomic analysis. The results showed that additional far-red light increased stem length and carbohydrate contents in multiple organs and decreased leaf area, specific leaf weight and dry weight of leaves in comparison with their counterparts grown under white light. Compared to white light, the maximum net photosynthetic rate of the leaves was increased by 31.72% by far-red light supplementation, indicating that far-red light indeed elevated the photosynthesis efficiency of grapes. Transcriptome analysis revealed that leaves were most responsive to far-red light, followed by sink organs, including stems and roots. Genes related to light signaling and carbon metabolites were tightly correlated with variations in the aforementioned physiological traits. In particular, VvLHCB1 is involved in light harvesting and restoring the balance of photosystem I and photosystem II excitation, and VvCOP1 and VvPIF3, which regulate light signal transduction, were upregulated under far-red conditions. In addition, the transcript abundances of the sugar transporter-encoding genes VvSWEET1 and VvSWEET3 and the carbon metabolite-encoding genes VvG6PD, VvSUS7 and VvPGAM varied in line with the change in sugar content.ConclusionsThis study showed that far-red light synergistically functioning with white light has a beneficial effect on grape photosystem activity and is able to differentially affect the growth of sink organs, providing evidence for the possible addition of far-red light to the wavelength range of photosynthetically active radiation (PAR).

Multidrug resistance of Botrytis cinerea associated with its adaptation to plant secondary metabolites.

Fungicides are an effective way to control gray mold of grapes, but the pathogen Botrytis cinerea can develop resistance, overcoming the effectiveness of a fungicide that is repeatedly applied. More importantly, the emergence of multidrug resistance (MDR) in the field, where multiple fungicides with different modes of action simultaneously lose their efficacies, is a significant concern. MDR is associated with ATP-binding cassette (ABC) transporters of the pathogen, and certain plant secondary metabolites (PSMs) stimulate the upregulation of ABC transporters, we hypothesized that the pathogen's preadaptation to PSMs might contribute to MDR development. To test this in B. cinerea, ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen, were selected based on their association with ABC transporters involved in fungicide resistance. B. cinerea strain B05.10 was continuously transferred for 15 generations on potato dextrose agar amended with a PSM (PDAP), and sensitivities to PSMs and fungicides were examined on the 5th, 10th, and 15th generations. RNA was extracted from B. cinerea from the selected generations. After 15 generations of culture transfers, an up-regulation was observed in the expression of ABC transporter-encoding genes BcatrB, BcatrD, and BcatrK using quantitative polymerase chain reaction (qPCR). This upregulation was found to contribute to MDR of B. cinerea against two or more fungicides, among azoxystrobin, boscalid, fludioxonil, difenoconazole, prochloraz, and pyrimethanil. This finding was confirmed through genetic transformation. The decreased sensitivity of B. cinerea to fungicides was confirmed as a subsequent MDR phenotype after exposure to camptothecin, flavanone, and resveratrol. Besides, transcriptome analysis also revealed the upregulation of transcription factors related to ABC expression following resveratrol exposure. This suggests that PSMs contributed to inducing preadaptation of B. cinerea, leading to subsequent MDR.IMPORTANCEThe emergence of MDR in plant pathogens is a threat to plant disease management and leads to the use of excessive fungicides. Botrytis cinerea is of particular concern because its MDR has widely emerged in the field. Understanding its genesis is the first step for controlling MDR. In this study, the contribution of PSMs to MDR has been examined. Effective management of this pathogen in agroecosystems relies on a better understanding of how it copes with phytochemicals or fungicides.

SLC35A2 expression drives breast cancer progression via ERK pathway activation.

Alterations in glycosylation are associated with breast tumor formation and progression. Nevertheless, the specific functions and mechanisms of the human major UDP-galactose transporter-encoding gene solute carrier family 35 member A2 (SLC35A2) in breast invasive carcinoma (BRCA) have not been fully determined. Here, we report that SLC35A2 promotes BRCA progression by activating extracellular signal regulated kinase (ERK). SLC35A2 expression and prognosis-predictive significance in pan-cancer were evaluated using public databases. The upstream non-coding RNAs (ncRNAs) of SLC35A2 were analyzed, and their expression and regulations were validated in breast tissues and cell lines by a quantitative PCR and dual-luciferase assays. We used bioinformatic tools to assess the link between SLC35A2 expression and immune infiltration and performed immunohistochemistry for validation. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, transwell, flow cytometer and western blotting were used to assess the proliferation, motility, cell cycle and apoptosis of BRCA cells in vitro. The xenograft models were constructed to assess the effect of SLC35A2 on BRCA tumor growth in vivo. The results indicated that SLC35A2 expression was upregulated and linked to an unfavorable prognosis in BRCA. The most likely upstream ncRNA-associated pathway of SLC35A2 in BRCA was the AC074117.1/hsa-let-7b-5p axis. SLC35A2 expression had positive correlations with the presence of Th2 cells, regulatory T cells and immune checkpoints. Knockdown of SLC35A2 could reduce BRCA cell proliferation, motility, and cause G2/M arrest and cell apoptosis via ERK signaling. Moreover, ERK activation can rescue the inhibitory effects of knockdown SLC35A2 in BRCA. In conclusion, AC074117.1/hsa-let-7b-5p axis-mediated high expression of SLC35A2 acts as a tumor promoter in BRCA via ERK signaling, which provides a potential target for BRCA treatment.

Sugar accumulation enhancement in sorghum stem is associated with reduced reproductive sink strength and increased phloem unloading activity.

Sweet sorghum has emerged as a promising source of bioenergy mainly due to its high biomass and high soluble sugar yield in stems. Studies have shown that loss-of-function Dry locus alleles have been selected during sweet sorghum domestication, and decapitation can further boost sugar accumulation in sweet sorghum, indicating that the potential for improving sugar yields is yet to be fully realized. To maximize sugar accumulation, it is essential to gain a better understanding of the mechanism underlying the massive accumulation of soluble sugars in sweet sorghum stems in addition to the Dry locus. We performed a transcriptomic analysis upon decapitation of near-isogenic lines for mutant (d, juicy stems, and green leaf midrib) and functional (D, dry stems and white leaf midrib) alleles at the Dry locus. Our analysis revealed that decapitation suppressed photosynthesis in leaves, but accelerated starch metabolic processes in stems. SbbHLH093 negatively correlates with sugar levels supported by genotypes (DD vs. dd), treatments (control vs. decapitation), and developmental stages post anthesis (3d vs.10d). D locus gene SbNAC074A and other programmed cell death-related genes were downregulated by decapitation, while sugar transporter-encoding gene SbSWEET1A was induced. Both SbSWEET1A and Invertase 5 were detected in phloem companion cells by RNA in situ assay. Loss of the SbbHLH093 homolog, AtbHLH093, in Arabidopsis led to a sugar accumulation increase. This study provides new insights into sugar accumulation enhancement in bioenergy crops, which can be potentially achieved by reducing reproductive sink strength and enhancing phloem unloading.

Multidrug resistance of Rhizoctonia solani determined by enhanced efflux for fungicides

Plant pathogens can develop multidrug resistance (MDR) through metabolomic and efflux activities. Although MDR has been observed in the field, its mechanisms are yet to be further studied. MDR in Rhizoctonia solani induced by the uncoupler SYP-14288, which involved efflux transporters including ATP binding cassette (ABC) and major facilitator superfamily (MFS) have been reported in our previous study. To confirm this, corresponding genes of the wild-type R. solani X19 and its derived MDR mutant X19–7 were compared through transcriptomics, RNA-Seq data validation, and heterologous expression. Genes encoding six ABC transporters and seven MFS transporters were identified to be associated with MDR and mostly showed a constitutively higher expression in X19–7 than in X19 regardless of SYP-14288 treatment. Eight ABC transporter-encoding genes and eight MFS transporter-encoding genes were further characterized by transferring into Saccharomyces cerevisiae. The sensitivity of transformants containing either ABC transporter-encoding gene AG1IA_06082 and MFS transporter-encoding gene AG1IA_08645 was significantly decreased in responses to fungicides having various modes of action including SYP-14288, fluazinam, chlorothalonil, and difenoconazole, indicating that these two genes were related to MDR. The roles of two genes were further confirmed by successfully detecting their protein products and high accumulation of SYP-14288 in yeast transformants. Thus, ABC and MFS transporters contributed to the development of MDR in R. solani. The result helps to understand the cause and mechanisms that influence the efficient use of fungicide.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202

BackgroundPolymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront.MethodsPandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling.ResultsThe MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites.ConclusionsThese findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Transporter Proteins as Ecological Assets and Features of Microbial Eukaryotic Pangenomes.

Here we review two connected themes in evolutionary microbiology: (a) the nature of gene repertoire variation within species groups (pangenomes) and (b) the concept of metabolite transporters as accessory proteins capable of providing niche-defining "bolt-on" phenotypes. We discuss the need for improved sampling and understanding of pangenome variation in eukaryotic microbes. We then review the factors that shape the repertoire of accessory genes within pangenomes. As part of this discussion, we outline how gene duplication is a key factor in both eukaryotic pangenome variation and transporter gene family evolution. We go on to outline how, through functional characterization of transporter-encoding genes, in combination with analyses of how transporter genes are gained and lost from accessory genomes, we can reveal much about the niche range, the ecology, and the evolution of virulence of microbes. We advocate for the coordinated systematic study of eukaryotic pangenomes through genome sequencing and the functional analysis of genes found within the accessory gene repertoire.

Double-Fermented Soybean Meal Totally Replaces Soybean Meal in Broiler Rations with Favorable Impact on Performance, Digestibility, Amino Acids Transporters and Meat Nutritional Value

Inclusion of microbial fermented soybean meal in broiler feed has induced advantageous outcomes for their performance and gastrointestinal health via exhibiting probiotic effects. In this study, soybean meal (SBM) was subjected to double-stage microbial fermentation utilizing functional metabolites of fungi and bacteria. In broiler diet, DFSBM replaced SBM by 0, 25, 50 and 100%. DFSBM was reported to have higher protein content and total essential, nonessential and free amino acids (increased by 3.67%, 12.81%, 10.10% and 5.88-fold, respectively, compared to SBM). Notably, phytase activity and lactic acid bacteria increased, while fiber, lipid and trypsin inhibitor contents were decreased by 14.05%, 38.24% and 72.80%, respectively, in a diet containing 100% DFSBM, compared to SBM. Improved growth performance and apparent nutrient digestibility, including phosphorus and calcium, and pancreatic digestive enzyme activities were observed in groups fed higher DFSBM levels. In addition, higher inclusion levels of DFSBM increased blood immune response (IgG, IgM, nitric oxide and lysozyme levels) and liver antioxidant status. Jejunal amino acids- and peptide transporter-encoding genes (LAT1, CAT-1, CAT-2, PepT-1 and PepT-2) were upregulated with increasing levels of DFSBM in the ration. Breast muscle crude protein, calcium and phosphorus retention were increased, especially at higher inclusion levels of DFSBM. Coliform bacteria load was significantly reduced, while lactic acid bacteria count in broiler intestines was increased with higher dietary levels of DFSBM. In conclusion, replacement of SBM with DFSBM positively impacted broiler chicken feed utilization and boosted chickens' amino acid transportation, in addition to improving the nutritional value of their breast meat.

Production of acetoin and its derivative tetramethylpyrazine from okara hydrolysate with Bacillus subtilis

Okara, a renewable biomass resource, is a promising fermentative raw material for the bio-production of value-added chemicals due to its abundance and low-costs. we developed a process for the enzymatic hydrolysis of okara, and then engineered Bacillus subtilis to utilize mixed sugars to produce acetoin in okara hydrolysis without the addition of a supplemental nitrogen source. Okara was initially hydrolyzed with cellulase, β-glucosidase, and pectinase to obtain okara hydrolysate containing mixed sugars (32.78 ± 0.23 g/L glucose, 1.43 ± 0.064 g/L arabinose, 7.74 ± 0.11 g/L galactose) and amino acids. In this study, Bacillus subtilis 168 was used as the acetoin-producing strain, and the key genes bdhA and acoA of the acetoin catabolism pathway were knocked out to improve the fermentation yield of acetoin. In order to utilize the galactose in the hydrolysate, the recombinant strain BS03 (Bacillus subtilis168∆bdhA∆acoA) was used to overexpress the arabinose transporter-encoding gene (araE) drive heterologous expression of the Leloir pathway gene (galKTE). The corn dry powder concentration was optimized to 29 g/L in the reducing sugar okara hydrolysate. The results show that the recombinant bacterium BS03 could still synthesize 11.79 g/L acetoin without using corn dry powder as a nitrogen source. Finally, using okara enzymatic hydrolysate as the carbon and nitrogen source, 11.11 g/L and 29.7 g/L acetoin were obtained by batch fermentation and fed-batch fermentation, respectively, which was further converted to 5.33 g/L and 13.37 g/L tetramethylpyrazine (TTMP) by reaction with an ammonium salt.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

Identification and expression analysis of sugar transporter family genes reveal the role of ZmSTP2 and ZmSTP20 in maize disease resistance

Sugar is an indispensable source of energy for plant growth and development, and it requires the participation of sugar transporter proteins (STPs) for crossing the hydrophobic barrier in plants. Here, we systematically identified the genes encoding sugar transporters in the genome of maize (Zea mays L.), analyzed their expression patterns under different conditions, and determined their functions in disease resistance. The results showed that the mazie sugar transporter family contained 24 members, all of which were predicted to be distributed on the cell membrane and had a highly conserved transmembrane transport domain. The tissue-specific expression of the maize sugar transporter genes was analyzed, and the expression level of these genes was found to be significantly different in different tissues. The analysis of biotic and abiotic stress data showed that the expression levels of the sugar transporter genes changed significantly under different stress factors. The expression levels of ZmSTP2 and ZmSTP20 continued to increase following Fusarium graminearum infection. By performing disease resistance analysis of zmstp2 and zmstp20 mutants, we found that after inoculation with Cochliobolus carbonum, Setosphaeria turcica, Cochliobolus heterostrophus, and F. graminearum, the lesion area of the mutants was significantly higher than that of the wild-type B73 plant. In this study, the genes encoding sugar transporters in maize were systematically identified and analyzed at the whole genome level. The expression patterns of the sugar transporter-encoding genes in different tissues of maize and under biotic and abiotic stresses were revealed, which laid an important theoretical foundation for further elucidation of their functions.

Antifungal activities of metconazole against the emerging wheat pathogen Fusarium pseudograminearum

Fusarium crown rot of wheat is a serious fungal disease that occurs worldwide. The disease has been emerging in the major wheat-growing areas in China since 2010. Fusarium pseudogramineaum is the predominant causative pathogen of crown rot of wheat in China. The 14α-demethylation inhibitor (DMI) fungicide metconazole has been shown to be effective against Fusarium spp., but little is known about its specific activity against F. pseudogramineaum. Metconazole exhibited strong antifungal activities against all thirty-nine F. pseudogramineaum strains collected from the major wheat-growing areas in China. Metconazole inhibited mycelial growth and conidial germ tube elongation of F. pseudograminearum. Metconazole treatment significantly reduced the production of major toxins and the expression levels of toxin biosynthesis genes. Genome-wide transcriptional profiling of F. pseudograminearum in response to metconazole indicated that the expression of genes involved in ergosterol biosynthesis, including fungicide target genes (cyp51 genes), was significantly induced by metconazole. Nine ATP-binding cassette (ABC) transporter-encoding genes were significantly expressed in response to metconazole treatment. Reduced ergosterol production and antioxidant enzyme activities were observed after metconazole treatment. Greenhouse experiments indicated a significant reduction in crown rot occurrence in wheat after seed treatment with metconazole. This study evaluated the potential of metconazole to manage wheat crown rot and provides information to understand its antifungal activities and mechanism of action against F. pseudograminearum.

BHLH010/089 Transcription Factors Control Pollen Wall Development via Specific Transcriptional and Metabolic Networks in Arabidopsis thaliana.

The pollen wall is a specialized extracellular cell wall that protects male gametophytes from various environmental stresses and facilitates pollination. Here, we reported that bHLH010 and bHLH089 together are required for the development of the pollen wall by regulating their specific downstream transcriptional and metabolic networks. Both the exine and intine structures of bhlh010 bhlh089 pollen grains were severely defective. Further untargeted metabolomic and transcriptomic analyses revealed that the accumulation of pollen wall morphogenesis-related metabolites, including polysaccharides, glyceryl derivatives, and flavonols, were significantly changed, and the expression of such metabolic enzyme-encoding genes and transporter-encoding genes related to pollen wall morphogenesis was downregulated in bhlh010 bhlh089 mutants. Among these downstream target genes, CSLB03 is a novel target with no biological function being reported yet. We found that bHLH010 interacted with the two E-box sequences at the promoter of CSLB03 and directly activated the expression of CSLB03. The cslb03 mutant alleles showed bhlh010 bhlh089–like pollen developmental defects, with most of the pollen grains exhibiting defective pollen wall structures.

Chemically defined elicitors activate priming in tomato seedlings

ABSTRACT Tomato (Solanum lycopersicum L.) is an important crop that possesses about 35,000 genes. The treatment of plants with elicitors or pathogen attacks causes a cascade of defense reactions. We investigated tomato responses to the BamFXTM solution containing Zn and Cu elicitors and report the results of comparative transcriptome analysis of tomato seeds treated with Zn and Cu elicitors. The seeds were treated with optimum concentrations of Bam-FX solutions and subjected to cold methanolic extraction methods to obtain the secondary metabolites produced within them at different time intervals post-Bam-FX treatment. The metabolite mixture was analyzed using gas chromatography-mass spectrometry (GCMS). In transcriptome sequencing, GO and KEGG analyses revealed that the majority of the DEGs in BamFx-treated tomato was associated with primary and secondary metabolism, plant hormone signal transduction, TF regulation, transport, and responses to stimuli.The secondary metabolites found in the BamFX treated tomato seedlings – Esters of Fumaric acid, Succinic acid etc. The transcript levels of most auxin transporter-encoding genes changed significantly in the BamFX-treated seedlings (e.g., Solyc01g007010.3, a RING-type E3 ubiquitin transferase). The gene Solyc07g061720.3 for Gibberellin 2-oxidase and the Phorbol-ester/DAG-type domain-containing protein (Solyc02g068680.1) associated with the intracellular signaling genes were found upregulated in the BamFx-treated seeds. The time-dependent effect of the BamFX (1:500 for 60 min) was found to be regulating Abscisic acid signaling pathway genes (Solyc09g015380.1). This study identified many candidate genes for future functional analyses and laid a theoretical foundation for an improved understanding of the molecular mechanisms involved in the BamFx treatment of tomatoes to improve stress resistance.