Impact of Transgenic Maize Ruifeng125 on Diversity and Dynamics of Bacterial Community in Rhizosphere Soil.

With the development of commercialized planting of genetically modified crops, their ecological security risks remain a key topic of public concern. Insect-resistant genetically modified maize, Ruifeng125, which expresses a fusion Bt protein (Cry1Ab-Cry2Aj), has obtained the application safety certificate issued by the Chinese government. To determine the effects of Ruifeng125 on the diversity and dynamics of bacterial communities, the accumulation and degradation pattern of the fusion Bt protein in the rhizosphere soil of transgenic maize were detected. Results showed that the contents of Bt protein varied significantly at different developmental stages, but after straw was returned to the field, over 97% of Bt proteins were degraded quickly at the early stages (≤10 d) and then they were degraded at a relatively slow rate. In addition, the variations in bacterial community diversity in the rhizosphere soil were detected by 16S ribosomal RNA (Rrna) high-throughput sequencing technology. A total of 44 phyla, 435 families, and 842 genera were obtained by 16S rRNA sequencing, among which Proteobacteria, Actinobacia, Acidobacter Acidobacterium, and Chloroflexi were the dominant taxa. At the same developmental stage, no significant differences in soil bacterial diversity were detected between Ruifeng125 and its non-transgenic control variety. Further analysis revealed that developmental stage, rather than the transgenic event, made the greatest contribution to the changes in soil microbial diversity. This research provides important information for evaluating the impacts of Bt crops on the soil microbiome and establishes a theoretical foundation for their environmental safety assessment.

Impact of Insect-Resistant Transgenic Maize 2A-7 on Diversity and Dynamics of Bacterial Communities in Rhizosphere Soil.

Artificial modification of Bacillus thuringiensis (Bt) proteins can effectively improve their resistance to target pests, but the effect of such modification on the diversity of rhizosphere microorganisms remains unclear. Transgenic maize 2A-7 contains two artificially modified Bt proteins, mCry1Ab and mCry2Ab. These proteins can enter soil and pose a potential threat to soil microbial diversity. To assess their impacts on rhizosphere bacteria communities, the contents of the two Bt proteins and changes in bacterial community diversity in the rhizosphere soils of transgenic maize 2A-7 and its control variety were analyzed at different growth stages in 2020. The results showed that the two Bt proteins were detected at low levels in the rhizosphere soils of 2A-7 plants. No significant differences in soil bacterial diversity were detected between 2A-7 and its control variety at any of the growth stages. Bioinformatics analysis indicated that the growth stage, rather than the cultivar, was the main factor causing changes in bacterial communities. This research provides valuable data for understanding the impact of Bt crops on the soil microbiome, and establishes a theoretical basis for evaluation of their safety.

Effects of Insect-Resistant Maize 2A-7 Expressing mCry1Ab and mCry2Ab on the Soil Ecosystem.

Transgenic maize 2A-7 expressing mCry1Ab and mCry2Ab has excellent resistance to lepidopteran pests. Previous studies have investigated the effects of several Bacillus thuringiensis (Bt) proteins on the soil. However, the effects of artificially modified Bt proteins on soil ecosystems are still unclear. To evaluate the effects of transgenic maize 2A-7 on soil, the physicochemical properties, enzyme activities and functional diversities of the microbial communities in rhizosphere soils from 2A-7 and its near-isogenic non-transgenic control Dongdan 6531 were analyzed at different developmental stages under field conditions. The alteration of six physicochemical properties (pH, total nitrogen, total phosphorus, organic matter, available phosphorus and alkali-hydrolyzed nitrogen) and six functional enzymes (catalase, alkaline phosphatase, sucrase, acid phosphatase, urease and alkaline protease) activities in the rhizosphere soils between the two maize cultivars were drastically correlated with plant growth stage, but not affected by the artificially modified Bt transgenes. An analysis of time-course Biolog data revealed that the functional diversity of microbial communities in the rhizosphere soil of 2A-7 and its control were similar at each developmental stage. The results suggest that transgenic maize 2A-7 has no significant impact on the soil ecosystem and provide valuable information on scientific safety assessments of 2A-7 and its commercial applications.

FgBUD14 is important for ascosporogenesis and involves both stage-specific alternative splicing and RNA editing during sexual reproduction.

In wheat head blight fungus Fusarium graminearum, A-to-I RNA editing occurs specifically during sexual reproduction. Among the genes with premature stop codons (PSCs) that require RNA editing to encode full-length proteins, FgBUD14 also had alternative splicing events in perithecia. In this study, we characterized the functions of FgBUD14 and its post-transcriptional modifications during sexual reproduction. The Fgbud14 deletion mutant was slightly reduced in growth, conidiation and virulence. Although deletion of FgBUD14 had no effect on perithecium morphology, the Fgbud14 mutant was defective in crozier formation and ascus development. The FgBud14-GFP localized to the apex of ascogenous hyphae and croziers, which may be related to its functions during early sexual development. During vegetative growth and asexual reproduction, FgBud14-GFP localized to hyphal tips and both ends of conidia. Furthermore, mutations blocking the splicing of intron 2 that has the PSC site had no effect on the function of FgBUD14 during sexual reproduction but caused a similar defect in growth with Fgbud14 mutant. Expression of the non-editable FgBUD14Intron2-TAA mutant allele also failed to complement the Fgbud14 mutant. Taken together, FgBUD14 plays important roles in ascus development, and both alternative splicing and RNA editing occur specifically to its transcripts during sexual reproduction in F. graminearum.

Stage-specific regulation of purine metabolism during infectious growth and sexual reproduction in Fusarium graminearum.

Ascospores generated during sexual reproduction are the primary inoculum for the wheat scab fungus Fusarium graminearum. Purine metabolism is known to play important roles in fungal pathogens but its lifecycle stage-specific regulation is unclear. By characterizing the genes involved in purine de novo and salvage biosynthesis pathways, we showed that de novo syntheses of inosine, adenosine and guanosine monophosphates (IMP, AMP and GMP) are important for vegetative growth, sexual/asexual reproduction, and infectious growth, whereas purine salvage synthesis is dispensable for these stages in F. graminearum. Addition of GMP rescued the defects of the Fgimd1 mutant in vegetative growth and conidiation but not sexual reproduction, whereas addition of AMP rescued all of these defects of the Fgade12 mutant, suggesting that the function of de novo synthesis of GMP rather than AMP is distinct in sexual stages. Moreover, Acd1, an ortholog of AMP deaminase, is dispensable for growth but essential for ascosporogenesis and pathogenesis, suggesting that AMP catabolism has stage-specific functions during sexual reproduction and infectious growth. The expression of almost all the genes involved in de novo purine synthesis is downregulated during sexual reproduction and infectious growth relative to vegetative growth. This study revealed that F. graminearum has stage-specific regulation of purine metabolism during infectious growth and sexual reproduction.

FgPal1 regulates morphogenesis and pathogenesis in Fusarium graminearum.

Ascospores are the primary inoculum in Fusarium graminearum, a causal agent of wheat head blight. In a previous study, FgPAL1 was found to be upregulated in the Fgama1 mutant and important for ascosporogenesis. However, the biological function of this well-conserved gene in filamentous ascomycetes is not clear. In this study, we characterized its functions in growth, differentiation and pathogenesis. The Fgpal1 mutant had severe growth defects and often displayed abnormal hyphal tips. It was defective in infectious growth in rachis tissues and spreading in wheat heads. The Fgpal1 mutant produced conidia with fewer septa and more nuclei per compartment than the wild type. In actively growing hyphal tips, FgPal1-GFP mainly localized to the subapical collar and septa. The FgPal1 and LifeAct partially co-localized at the subapical region in an interdependent manner. The Fgpal1 mutant was normal in meiosis with eight nuclei in developing asci but most asci were aborted. Taken together, our results showed that FgPal1 plays a role in maintaining polarized tip growth and coordination between nuclear division and cytokinesis, and it is also important for infectious growth and developments of ascospores by the free cell formation process.

An expanded subfamily of G-protein-coupled receptor genes in Fusarium graminearum required for wheat infection.

The cAMP-PKA and MAP kinase pathways are essential for plant infection in the wheat head blight fungus Fusarium graminearum. To identify upstream receptors of these well-conserved signalling pathways, we systematically characterized the 105 G-protein-coupled receptor (GPCR) genes. Although none were required for vegetative growth, five GPCR genes (GIV1-GIV5) significantly upregulated during plant infection were important for virulence. The giv1 mutant was defective in the formation of specialized infection structures known as infection cushions, which was suppressed by application of exogenous cAMP and dominant active FST7 MEK kinase. GIV1 was important for the stimulation of PKA and Gpmk1 MAP kinase by compounds in wheat spikelets. GIV2 and GIV3 were important for infectious growth after penetration. Invasive hyphae of the giv2 mutant were defective in cell-to-cell spreading and mainly grew intercellularly in rachis tissues. Interestingly, the GIV2-GIV5 genes form a phylogenetic cluster with GIV6, which had overlapping functions with GIV5 during pathogenesis. Furthermore, the GIV2-GIV6 cluster is part of a 22-member subfamily of GPCRs, with many of them having in planta-specific upregulation and a common promoter element; however, only three subfamily members are conserved in other fungi. Taken together, F. graminearum has an expanded subfamily of infection-related GPCRs for regulating various infection processes.

The meiosis-specific APC activator FgAMA1 is dispensable for meiosis but important for ascosporogenesis in Fusarium graminearum.

Ascospores are the primary inoculum in Fusarium graminearum. Interestingly, 70 of its genes have premature stop codons (PSC) and require A-to-I editing during sexual reproduction to encode full-length proteins, including the ortholog of yeast Ama1, a meiosis-specific activator of APC/C. In this study, we characterized the function of FgAMA1 and its PSC editing. FgAMA1 was specifically expressed during sexual reproduction. The Fgama1 mutant was normal in growth and perithecium formation but defective in ascospogenesis. Instead of forming four-celled, uninucleate ascospores, Fgama1 mutant produced oval, single-celled, binucleated ascospores by selfing. Some mutant ascospores began to bud and underwent additional mitosis inside asci. Expression of the wild-type or edited FgAMA1 but not the uneditable allele complemented Fgama1. In the Fgama1 x mat-1-1 outcross, over 60% of the asci had eight Fgama1 or intermediate (elongated but single-celled) ascospores, suggesting efficient meiotic silencing of unpaired FgAMA1. Deletion of FgPAL1, one of the genes upregulated in Fgama1 also resulted in defects in ascospore morphology and budding. Overall, our results showed that FgAMA1 is dispensable for meiosis but important for ascospore formation and discharge. In F. graminearum, whereas some of its targets are functional during meiosis, FgAma1 may target other proteins that function after spore delimitation.

Sexual specific functions of Tub1 beta-tubulins require stage-specific RNA processing and expression in Fusarium graminearum.

The wheat head blight fungus Fusarium graminearum has two highly similar beta-tubulin genes with overlapping functions during vegetative growth but only TUB1 is important for sexual reproduction. To better understand their functional divergence during ascosporogenesis, in this study we characterized the sequence elements important for stage-specific functions of TUB1. Deletion of TUB1 blocked the late but not initial stages of perithecium formation. Perithecia formed by tub1 mutant had limited ascogenous hyphae and failed to develop asci. Silencing of TUB1 by MSUD also resulted in defects in ascospore formation. Interestingly, the 3'-UTR of TUB1 was dispensable for growth but essential for its function during sexual reproduction. RIP mutations that specifically affected Tub1 functions during sexual reproduction also were identified in two ascospore progeny. Furthermore, site-directed mutagenesis showed that whereas the non-editable mutations at three A-to-I RNA editing sites had no effects, the N347D (not T362D or I368V) edited mutation affected ascospore development. In addition, the F167Y, but not E198K or F200Y, mutation in TUB1 conferred tolerance to carbendazim and caused a minor defect in sexual reproduction. Taken together, our data indicate TUB1 plays an essential role in ascosporogenesis and sexual-specific functions of TUB1 require stage-specific RNA processing and Tub1 expression.

RNA editing of the AMD1 gene is important for ascus maturation and ascospore discharge in Fusarium graminearum.

Ascospores are the primary inoculum in the wheat scab fungus Fusarium graminearum that was recently shown to have sexual stage-specific A-to-I RNA editing. One of the genes with premature-stop-codons requiring A-to-I editing to encode full-length functional proteins is AMD1 that encodes a protein with a major facilitator superfamily (MFS) domain. Here, we characterized the functions of AMD1 and its UAG to UGG editing event. The amd1 deletion mutant was normal in growth and conidiation but defective in ascospore discharge due to the premature breakdown of its ascus wall in older perithecia, which is consistent with the specific expression of AMD1 at later stages of sexual development. Expression of the wild-type or edited allele of AMD1 but not un-editable allele rescued the defects of amd1 in ascospore discharge. Furthermore, Amd1-GFP localized to the ascus membrane and Amd1 orthologs are only present in ascocarp-forming fungi that physically discharge ascospores. Interestingly, deletion of AMD1 results in the up-regulation of a number of genes related to transporter activity and membrane functions. Overall, these results indicated that Amd1 may play a critical role in maintaining ascus wall integrity during ascus maturation, and A-to-I editing of its transcripts is important for ascospore discharge in F. graminearum.

FgSsn3 kinase, a component of the mediator complex, is important for sexual reproduction and pathogenesis in Fusarium graminearum.

Fusarium graminearum is an important pathogen of wheat and barley. In addition to severe yield losses, infested grains are often contaminated with harmful mycotoxins. In this study, we characterized the functions of FgSSN3 kinase gene in different developmental and infection processes and gene regulation in F. graminearum. The FgSSN3 deletion mutant had a nutrient-dependent growth defects and abnormal conidium morphology. It was significantly reduced in DON production, TRI gene expression, and virulence. Deletion of FgSSN3 also resulted in up-regulation of HTF1 and PCS1 expression in juvenile cultures, and repression of TRI genes in DON-producing cultures. In addition, Fgssn3 was female sterile and defective in hypopodium formation and infectious growth. RNA-seq analysis showed that FgSsn3 is involved in the transcriptional regulation of a wide variety genes acting as either a repressor or activator. FgSsn3 physically interacted with C-type cyclin Cid1 and the cid1 mutant had similar phenotypes with Fgssn3, indicating that FgSsn3 and Cid1 form the CDK-cyclin pair as a component of the mediator complex in F. graminearum. Taken together, our results indicate that FgSSN3 is important for secondary metabolism, sexual reproduction, and plant infection, as a subunit of mediator complex contributing to transcriptional regulation of diverse genes.

Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes.

Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1(FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA(1831)GUA(1834)G, in its kinase domain were changed to UG(1831)GUG(1834)G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG(1831)GUG(1834)G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites inF. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69PUK1-like pseudogenes with stop codons in ORFs.PUK1orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides Furthermore,F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing inF. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs.

FgCDC14 regulates cytokinesis, morphogenesis, and pathogenesis in Fusarium graminearum.

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk-type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter-connected conidia. In the interphase, FgCdc14-GFP localized to the nucleus and spindle-pole-body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.