Presentation_1_Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.pptx

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.</p

Genome-wide identification and characterization of GATA transcription factor gene family in Alternaria alternata

GATA transcription factors (TFs) comprise a protein family whose members contain either one or two highly conserved zinc finger DNA-binding domains, have been shown to play a critical role in regulating various biological processes. Previous studies have shown that the GATA TF AaSreA plays important role in iron homeostasis and oxidative stress resistance of Alternaria alternata. In the present study, we systematically identified additional five GATA TFs (AaAreA, AaAreB, AaLreA, AaLreB, AaNsdD) and characterized their functions in Alternaria alternata. Our results indicated that AaAreB is only crucial for vegetative growth while AaAreA, AaLreB, and AaNsdD positively regulate vegetative growth, conidiation as well pathogenicity. Interestingly, all AaGATA TFs are important for ROS detoxification. AaAreA is the most important in the pathogenic process and the RNA-seq revealed that AaAreA contributes to many other biological processes including ribosome biogenesis, amino acid metabolism and secondary metabolite biogenesis. Taking together, in addition to AaSreA previously reported, other five GATA proteins are also involved in a complex process governing development, conidiation, ROS detoxification, and pathogenicity in A. alternata.</p

Data_Sheet_1_Cell-Wall-Degrading Enzymes Required for Virulence in the Host Selective Toxin-Producing Necrotroph Alternaria alternata of Citrus.xlsx

The necrotrophic fungal pathogen Alternaria alternata attacks many citrus species, causing brown spot disease. Its pathogenic capability depends primarily on the production of host-selective ACT toxin. In the current study a Ste12 transcription factor was characterized to be required for conidial formation and the production of cell-wall-degrading enzymes (CWDEs) in the tangerine pathotype of A. alternata. The Ste12 deficiency strain (ΔSte12) retained wild-type growth, ACT toxin production, and sensitivity to oxidative and osmotic stress. However, pathogenicity tests assayed on detached Dancy leaves revealed a marked reduction in virulence of ΔSte12. Transcriptome and quantitative RT-PCR analyses revealed that many genes associated with Carbohydrate-Active Enzymes (CAZymes) were downregulated in ΔSte12. Two cutinase-coding genes (AaCut3 and AaCut7) regulated by Ste12 were individually and simultaneously inactivated. The AaCut3 or AaCut7 deficiency strain unchanged in cutinase activities and incited wild-type lesions on Dancy leaves. However, the strain carrying an AaCut3 AaCut7 double mutation produced and secreted significantly fewer cutinases and incited smaller necrotic lesions than wild type. Not only is the host-selective toxin (HST) produced by A. alternata required for fungal penetration and lesion formation, but so too are CWDEs required for full virulence. Overall, this study expands our understanding of how A. alternata overcomes citrus physical barriers to carry out successful penetration and colonization.</p

Table_4_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.xlsx

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

DataSheet_1_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.pdf

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

Table_1_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.xlsx

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

Table_2_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.xlsx

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

Table_3_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.xlsx

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

Table_5_Nanopore long-read RNAseq reveals transcriptional variations in citrus species.xlsx

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5’, 3’, or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.</p

Kaplan-Meier survival curves for NSCLC patients according to serum level of miR-429.

<p><i>P</i>-value for survival of patients with high and low levels of miRNA expression was calculated using the log-rank test. *<i>P</i><0.05 between groups.</p