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In-Depth Transcriptome Sequencing of Mexican Lime Trees Infected with Candidatus Phytoplasma aurantifolia.

Mardi M, Karimi Farsad L, Gharechahi J, Salekdeh GH - PLoS ONE (2015)

Bottom Line: When the abundances of unigenes in healthy and infected plants were compared, 2,805 transcripts showed significant differences (false discovery rate ≤ 0.001 and log2 ratio ≥ 1.5).These differentially expressed genes (DEGs) were significantly enriched in 43 KEGG metabolic and regulatory pathways.The findings can be valuable for unravelling the molecular mechanisms of plant-phytoplasma interactions and can pave the way for engineering lime trees with resistance to witches' broom disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Tehran, Iran.

ABSTRACT
Witches' broom disease of acid lime greatly affects the production of Mexican lime in Iran. It is caused by a phytoplasma (Candidatus Phytoplasma aurantifolia). However, the molecular mechanisms that underlie phytoplasma pathogenicity and the mode of interactions with host plants are largely unknown. Here, high-throughput transcriptome sequencing was conducted to explore gene expression signatures associated with phytoplasma infection in Mexican lime trees. We assembled 78,185 unique transcript sequences (unigenes) with an average length of 530 nt. Of these, 41,805 (53.4%) were annotated against the NCBI non-redundant (nr) protein database using a BLASTx search (e-value ≤ 1e-5). When the abundances of unigenes in healthy and infected plants were compared, 2,805 transcripts showed significant differences (false discovery rate ≤ 0.001 and log2 ratio ≥ 1.5). These differentially expressed genes (DEGs) were significantly enriched in 43 KEGG metabolic and regulatory pathways. The up-regulated DEGs were mainly categorized into pathways with possible implication in plant-pathogen interaction, including cell wall biogenesis and degradation, sucrose metabolism, secondary metabolism, hormone biosynthesis and signalling, amino acid and lipid metabolism, while down-regulated DEGs were predominantly enriched in ubiquitin proteolysis and oxidative phosphorylation pathways. Our analysis provides novel insight into the molecular pathways that are deregulated during the host-pathogen interaction in Mexican lime trees infected by phytoplasma. The findings can be valuable for unravelling the molecular mechanisms of plant-phytoplasma interactions and can pave the way for engineering lime trees with resistance to witches' broom disease.

No MeSH data available.


Related in: MedlinePlus

Quantitative real-time RT-PCR (qRT-PCR) confirmation of the differential expression of 25 DEGs in diseased and control plants (U76002; Ammonium transporter, U17862; WRKY transcription factor 21, U75775; Nitrite reductase, U2265; Amino acid transporter, U26576; Mitogen-activated protein kinase kinase kinase 1, U27316; NAC domain-containing protein 71, U18376; Cytochrome P450 84A1, U14388; DELLA protein RGL2, U3869; Jasmonate ZIM domain-containing protein 6, U59125; CRT/DRE binding factor, U35073; Allene oxide synthase, chloroplastic, U72184; Zinc finger A20 and AN1 domain-containing stress-associated protein 3, U17606; BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1, U59280; Miraculin-like protein 2, U352; Beta-galactosidase 3, U59467; Proline-rich protein, U17275; phytochrome-interacting factor 3, U24969; Probable LRR receptor-like serine/threonine-protein kinase, U77887; Gibberellin 2-oxidase, U68165; Ent-copalyl diphosphate synthase, U41653; LRR receptor-like serine/threonine-protein kinase GSO1, U35689; Chalcone synthase, U68593; Cyclic nucleotide-gated ion channel 1, U49255; Ent-kaurene oxidase, U17546; Leucine-rich repeat (LRR) protein).The signal intensity of each unigene was normalised against 18S rRNA as a housekeeping gene. The log2 fold change in expression of each transcript was determined using the 2-ΔΔCt method and is plotted for comparison with the log2 ratios determined using RPKM values in the RNA-seq data. Error bars represent standard error, which is calculated by dividing standard deviation to square root of N. N refers to number of replicate.
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pone.0130425.g004: Quantitative real-time RT-PCR (qRT-PCR) confirmation of the differential expression of 25 DEGs in diseased and control plants (U76002; Ammonium transporter, U17862; WRKY transcription factor 21, U75775; Nitrite reductase, U2265; Amino acid transporter, U26576; Mitogen-activated protein kinase kinase kinase 1, U27316; NAC domain-containing protein 71, U18376; Cytochrome P450 84A1, U14388; DELLA protein RGL2, U3869; Jasmonate ZIM domain-containing protein 6, U59125; CRT/DRE binding factor, U35073; Allene oxide synthase, chloroplastic, U72184; Zinc finger A20 and AN1 domain-containing stress-associated protein 3, U17606; BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1, U59280; Miraculin-like protein 2, U352; Beta-galactosidase 3, U59467; Proline-rich protein, U17275; phytochrome-interacting factor 3, U24969; Probable LRR receptor-like serine/threonine-protein kinase, U77887; Gibberellin 2-oxidase, U68165; Ent-copalyl diphosphate synthase, U41653; LRR receptor-like serine/threonine-protein kinase GSO1, U35689; Chalcone synthase, U68593; Cyclic nucleotide-gated ion channel 1, U49255; Ent-kaurene oxidase, U17546; Leucine-rich repeat (LRR) protein).The signal intensity of each unigene was normalised against 18S rRNA as a housekeeping gene. The log2 fold change in expression of each transcript was determined using the 2-ΔΔCt method and is plotted for comparison with the log2 ratios determined using RPKM values in the RNA-seq data. Error bars represent standard error, which is calculated by dividing standard deviation to square root of N. N refers to number of replicate.

Mentions: In order to evaluate technical and biological variations in our RNA-seq data and to validate our assembly, we further confirmed the differential expression of 25 DEG transcripts (12 down-regulated and 13 up-regulated unigenes) using qRT-PCR analysis. The results of qRT-PCR and the RNA-seq analyses for the selected DEGs are shown in Fig 4. Interestingly, all tested DEGs produced unique PCR products further confirming our assembly approach. In addition, all DEGs showed similar trends in terms of expression, except for U72184 and U17606, the trends of which differed between the two datasets. However, some of the DEGs including U76002, U27316, U18376, U3869, U352, U59467, U17275, U68165, U68593, and U17546 showed significant discrepancies between the qRT-PCR expression values and the RPKM values estimated from RNA-seq data. These discrepancies might have resulted from differences in the mathematical equations used for calculation of the corresponding expression values.


In-Depth Transcriptome Sequencing of Mexican Lime Trees Infected with Candidatus Phytoplasma aurantifolia.

Mardi M, Karimi Farsad L, Gharechahi J, Salekdeh GH - PLoS ONE (2015)

Quantitative real-time RT-PCR (qRT-PCR) confirmation of the differential expression of 25 DEGs in diseased and control plants (U76002; Ammonium transporter, U17862; WRKY transcription factor 21, U75775; Nitrite reductase, U2265; Amino acid transporter, U26576; Mitogen-activated protein kinase kinase kinase 1, U27316; NAC domain-containing protein 71, U18376; Cytochrome P450 84A1, U14388; DELLA protein RGL2, U3869; Jasmonate ZIM domain-containing protein 6, U59125; CRT/DRE binding factor, U35073; Allene oxide synthase, chloroplastic, U72184; Zinc finger A20 and AN1 domain-containing stress-associated protein 3, U17606; BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1, U59280; Miraculin-like protein 2, U352; Beta-galactosidase 3, U59467; Proline-rich protein, U17275; phytochrome-interacting factor 3, U24969; Probable LRR receptor-like serine/threonine-protein kinase, U77887; Gibberellin 2-oxidase, U68165; Ent-copalyl diphosphate synthase, U41653; LRR receptor-like serine/threonine-protein kinase GSO1, U35689; Chalcone synthase, U68593; Cyclic nucleotide-gated ion channel 1, U49255; Ent-kaurene oxidase, U17546; Leucine-rich repeat (LRR) protein).The signal intensity of each unigene was normalised against 18S rRNA as a housekeeping gene. The log2 fold change in expression of each transcript was determined using the 2-ΔΔCt method and is plotted for comparison with the log2 ratios determined using RPKM values in the RNA-seq data. Error bars represent standard error, which is calculated by dividing standard deviation to square root of N. N refers to number of replicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489016&req=5

pone.0130425.g004: Quantitative real-time RT-PCR (qRT-PCR) confirmation of the differential expression of 25 DEGs in diseased and control plants (U76002; Ammonium transporter, U17862; WRKY transcription factor 21, U75775; Nitrite reductase, U2265; Amino acid transporter, U26576; Mitogen-activated protein kinase kinase kinase 1, U27316; NAC domain-containing protein 71, U18376; Cytochrome P450 84A1, U14388; DELLA protein RGL2, U3869; Jasmonate ZIM domain-containing protein 6, U59125; CRT/DRE binding factor, U35073; Allene oxide synthase, chloroplastic, U72184; Zinc finger A20 and AN1 domain-containing stress-associated protein 3, U17606; BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1, U59280; Miraculin-like protein 2, U352; Beta-galactosidase 3, U59467; Proline-rich protein, U17275; phytochrome-interacting factor 3, U24969; Probable LRR receptor-like serine/threonine-protein kinase, U77887; Gibberellin 2-oxidase, U68165; Ent-copalyl diphosphate synthase, U41653; LRR receptor-like serine/threonine-protein kinase GSO1, U35689; Chalcone synthase, U68593; Cyclic nucleotide-gated ion channel 1, U49255; Ent-kaurene oxidase, U17546; Leucine-rich repeat (LRR) protein).The signal intensity of each unigene was normalised against 18S rRNA as a housekeeping gene. The log2 fold change in expression of each transcript was determined using the 2-ΔΔCt method and is plotted for comparison with the log2 ratios determined using RPKM values in the RNA-seq data. Error bars represent standard error, which is calculated by dividing standard deviation to square root of N. N refers to number of replicate.
Mentions: In order to evaluate technical and biological variations in our RNA-seq data and to validate our assembly, we further confirmed the differential expression of 25 DEG transcripts (12 down-regulated and 13 up-regulated unigenes) using qRT-PCR analysis. The results of qRT-PCR and the RNA-seq analyses for the selected DEGs are shown in Fig 4. Interestingly, all tested DEGs produced unique PCR products further confirming our assembly approach. In addition, all DEGs showed similar trends in terms of expression, except for U72184 and U17606, the trends of which differed between the two datasets. However, some of the DEGs including U76002, U27316, U18376, U3869, U352, U59467, U17275, U68165, U68593, and U17546 showed significant discrepancies between the qRT-PCR expression values and the RPKM values estimated from RNA-seq data. These discrepancies might have resulted from differences in the mathematical equations used for calculation of the corresponding expression values.

Bottom Line: When the abundances of unigenes in healthy and infected plants were compared, 2,805 transcripts showed significant differences (false discovery rate ≤ 0.001 and log2 ratio ≥ 1.5).These differentially expressed genes (DEGs) were significantly enriched in 43 KEGG metabolic and regulatory pathways.The findings can be valuable for unravelling the molecular mechanisms of plant-phytoplasma interactions and can pave the way for engineering lime trees with resistance to witches' broom disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Agricultural Biotechnology Research Institute of Iran, Karaj, Tehran, Iran.

ABSTRACT
Witches' broom disease of acid lime greatly affects the production of Mexican lime in Iran. It is caused by a phytoplasma (Candidatus Phytoplasma aurantifolia). However, the molecular mechanisms that underlie phytoplasma pathogenicity and the mode of interactions with host plants are largely unknown. Here, high-throughput transcriptome sequencing was conducted to explore gene expression signatures associated with phytoplasma infection in Mexican lime trees. We assembled 78,185 unique transcript sequences (unigenes) with an average length of 530 nt. Of these, 41,805 (53.4%) were annotated against the NCBI non-redundant (nr) protein database using a BLASTx search (e-value ≤ 1e-5). When the abundances of unigenes in healthy and infected plants were compared, 2,805 transcripts showed significant differences (false discovery rate ≤ 0.001 and log2 ratio ≥ 1.5). These differentially expressed genes (DEGs) were significantly enriched in 43 KEGG metabolic and regulatory pathways. The up-regulated DEGs were mainly categorized into pathways with possible implication in plant-pathogen interaction, including cell wall biogenesis and degradation, sucrose metabolism, secondary metabolism, hormone biosynthesis and signalling, amino acid and lipid metabolism, while down-regulated DEGs were predominantly enriched in ubiquitin proteolysis and oxidative phosphorylation pathways. Our analysis provides novel insight into the molecular pathways that are deregulated during the host-pathogen interaction in Mexican lime trees infected by phytoplasma. The findings can be valuable for unravelling the molecular mechanisms of plant-phytoplasma interactions and can pave the way for engineering lime trees with resistance to witches' broom disease.

No MeSH data available.


Related in: MedlinePlus