Transcriptome analysis of the differential effect of the NADPH oxidase gene RbohB in Phaseolus vulgaris roots following Rhizobium tropici and Rhizophagus irregularis inoculation

- differentially expressed genes in the PvRbohB-RNAi roots inoculated with Rhizobium tropici or Rhizophagus irregularis..
- We found that, in the early stages, root nodule symbioses generated larger changes of the transcriptome than did AM symbioses in P.
- Genes related to ROS homeostasis and cell wall flexibility were markedly upregulated in the early stages of rhizobial colonization, but not during AM colonization.
- Compared with AM colonization, the rhizobia induced the expression of a greater number of genes encoding enzymes involved in the metabolism of auxins, cytokinins, and ethylene, which were typically repressed in the PvRbohB-RNAi roots..
- Conclusions: Our research provides substantial insights into the genetic interaction networks in the early stages of rhizobia and AM symbioses with P.
- The mutual recognition of both the macro- and micro-symbionts leads to the plant- orchestrated formation of the fungal hypopodium on the surface of the root epidermis and the prepenetration ap- paratus in the underlying epidermal cell, which forms the entry route of the microsymbiont.
- calcium spiking in the root cells of Medicago truncatula, which is likely required for the formation of the pre-IT and the prepenetration apparatus, respectively [6].
- The activation of calcium spiking in the epidermal cells re- quires the enzyme 3-HYDROXY-3-METHYLGLUTARYL CoA REDUCTASE1, a key regulator of the mevalonate pathway reported to interact with the plasma membrane receptor-like kinase SYMBIOSIS RECEPTOR KINASE/.
- Nucleo- porins and cationic channels located in the nuclear enve- lope are also part of the common symbiotic pathways [12].
- The RbohE promoter is active in the arbuscule-hosting cells of M.
- vulgaris gene expression, the transcriptomes of the control (nonsilenced transgenic roots) and PvRbohB-RNAi roots inoculated with rhizobia (R.
- The data were deposited in the NCBI databases under the BioProject accession num- ber PRJNA482464..
- A total of 1402 and 278 were upregulated differ- entially expressed genes (DEGs) in the rhizobial and AM roots, respectively, of which 52 upregulated DEGs were shared between both datasets (Fig.
- Only 16 genes were found to be differentially regulated in the two symbioses.
- These results suggest that these genes could play important differential roles in the early stages of nodulation and mycorrhization.
- These same categories were less represented in the upregulated GO terms under mycorrhi- zation conditions.
- In the CC category, both the up- and downregulated genes under mycorrhiza- tion conditions constituted three main groups: nucleus, endoplasmic reticulum, and plasma membrane.
- Thus, GO term analysis revealed that the vast genetic reprograming observed in the early stages of nodulation and the more moderate changes observed dur- ing early mycorrhization largely involved genes associated.
- 1 MAPlots of the transcriptomes of control and PvRbohB-RNAi P.
- Several peroxidases and ethylene-related genes were induced in the PvRbohB-RNAi roots, suggesting a possible increase in the ROS and ethylene levels of these plants.
- PvRbohB silencing repressed the expression of genes involved in cell wall remodeling, such as CELLU- LOSE SYNTHASE and XYLOGLUCAN ENDOTRANS- GLUCOSYLASE/HYDROLASE, together with important genes in the cell cycle and auxin biosynthesis, such as the gene encoding THE INDOLE-3-PYRUVATE MONO- OXYGENASE YUCCA5 (Fig.
- Furthermore, a global functional annotation of the DEGs using GO terms indi- cated an induction in the expression of genes involved in biological regulation and catabolic processes, those.
- Figure S3), suggesting that PvRbohB plays a role in the signaling and gene regulation processes of P.
- In response to rhizo- bial inoculation, 1402 genes were upregulated in the control roots.
- however, only 293 of these genes were also induced in the inoculated PvRbohB-RNAi roots (Fig.
- Similarly, in mycorrhized roots, of the 278 genes up- regulated in the control transgenic roots, only two were induced in PvRbohB-RNAi roots (Fig.
- Further- more, 42 of the genes upregulated during mycorrhiza- tion in the control roots were downregulated in the PvRbohB-RNAi roots (Fig.
- b-e Venn diagrams indicate the total number of DEGs (b), and the numbers of upregulated (Up) (c), downregulated (Down) (d), and overlapping (e) genes in the rhizobia-inoculated and mycorrhizal roots.
- The DEGs were identified using a cutoff threshold of Log2FC ≥ 1.5 and a P-adj/FDR ≤ 0.05 in the DESeq, EdgeR, and NOISeq packages of.
- The normal transcriptional repression of a large set of genes during both symbiotic processes was substantially altered in the PvRbohB-RNAi roots.
- of the downregulated genes in the rhizobial-inoculated control roots were not downregulated in the PvRbohB- silenced roots at 7 dpi (Fig.
- of the 262 downregulated genes in the mycorrhized con- trol roots were similarly downregulated in the PvRbohB- RNAi roots, while an additional 201 genes were down- regulated in these transgenic plants, suggesting that the early stages of AM symbiosis were strongly impacted by PvRbohB silencing.
- Under nodulation conditions, 57 of the upregulated genes in the PvRbohB-RNAi roots were downregulated in the control roots.
- The PvRbohB-RNAi roots were found to have an 80% reduc- tion in the transcript level of this gene relative to the control, supporting the resulting phenotype.
- 3 Global analysis of the DEGs in PvRbohB-RNAi P.
- the RT-qPCR results support the findings obtained in the RNA-Seq analysis..
- In this study, we found that the expression levels of 28 ROS- scavenging genes were increased in the control roots.
- In the PvRbohB-silenced roots, how- ever, only 12 peroxidase genes were upregulated.
- however, the expression levels of these genes were un- affected in the PvRbohB-RNAi roots (Fig.
- Hydroxyl radicals are involved in the loosening of cell walls via an apoplastic peroxidase-dependent mech- anism, and hydrogen peroxide is involved in cell wall lig- nification [31, 32].
- 4 Global analysis of the DEGs in rhizobia-inoculated and mycorrhized PvRbohB-RNAi roots.
- 5b), reflecting the differ- ences in the developmental programs and kinetics of these symbioses.
- Under nodulation conditions, genes involved in cell wall biogenesis were induced in the PvRbohB-RNAi roots.
- however, some of these were downregulated in the control roots.
- DEGs encoding proteins related to ROS metabolism (a) and cell wall biosynthesis (b) in the control and PvRbohB-RNAi P.
- We identified 15 upregulated and 6 downregulated auxin-related genes in the rhizobia- inoculated control roots, which were mainly early auxin re- sponse and regulatory genes (Fig.
- The five upregulated early auxin signaling genes in the mycorrhized control roots differed from the auxin-induced genes in the nodulated control roots, indicating a differential regulation of the auxin-related genes in these symbiotic processes (Fig.
- However, in the PvRbohB-RNAi roots, the auxin pathway genes were induced in rhizobia-inoculated roots and down- regulated in mycorrhized control roots (Additional file 13:.
- In the control P.
- however, none of these genes were induced in the PvRbohB-silenced roots (Fig.
- In the mycorrhized control roots, the induction of genes related to cytokinin signaling was not observed, although two such genes were repressed.
- By contrast, two genes (UDP-GLYCOSYL- TRANSFERASE 83A1 and GLUCOSYL/GLUCURONOSYL TRANSFERASE) involved in cytokinin metabolism were upregulated in the PvRbohB-RNAi roots during mycorrhi- zation (Fig.
- downregulated genes related to the response and biosyn- thesis of ethylene in the control rhizobia-inoculated roots (Fig.
- In the PvRbohB-RNAi roots, the genes involved in the biosynthesis and response of ethylene were not induced to the same extent as in the control roots, suggesting that less ethylene production and a smaller ethylene response takes place in the PvRbohB-si- lenced roots during rhizobial symbioses (Fig.
- Simi- larly, genes involved in the ethylene response pathway were downregulated or not induced in PvRbohB-RNAi roots inoculated with AM (Fig.
- These results suggest that PvRbohB plays a crucial role in the ethylene responses of P.
- To validate the transcriptome results, we determined the transcript abundance of 9 DEGs in the control and PvRbohB-RNAi roots under nodulation and mycorrhiza- tion conditions using RT-qPCR.
- For mycorrhization, we evalu- ated one upregulated gene identified in the RNA-Seq ana- lysis, EF-HAND CALCIUM-BINDING DOMAIN- CONTAINING PROTEIN (EF-HANDM) (Fig.
- The PvENOD40 and PvNIN transcript levels were significantly increased in the control rhizobia- inoculated roots (Fig.
- however, these nodulins were not induced in the PvRbohB-RNAi roots, supporting our previous findings [13, 18].
- In the control roots colonized by AM, the PvEF-HANDM transcripts were significantly upregulated but were remarkably downregulated in the PvRbohB-RNAi roots (Fig.
- Furthermore, under nodulation conditions, PEROXIDASE1 (PvPO1), CARBOXYLESTERASE17 (PvCES17), XYLOGLU- CAN:XYLOGLUCOSYLTRANSFERASE (PvXGT), PvSAUR, and AMINOCYCLOPROPANE CARBOXYLATE OXIDASE (PvACCO) were upregulated in the control roots and down- regulated in the PvRbohB-RNAi roots (Fig.
- Simi- larly, PvCES17, PvXGT, PvSAUR, PvACCO, and ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR1 (PvERF1) were upregulated in the mycorrhized control roots but downregu- lated in the mycorrhized PvRbohB-silenced roots (Fig.
- 6 Heatmap of the expression patterns of genes involved in phytohormone metabolism.
- DEGs related to the auxin (a), cytokinin (b), and ethylene (c) signaling pathways in the control and PvRbohB-RNAi P.
- The lag in the kinetics of these symbiotic processes could partially ex- plain this difference.
- 7 Validation of the expression patterns of genes induced in control and PvRbohB-RNAi roots.
- contrast, assigned to the downregulated genes in the mycorrhized roots, suggesting a differential effect for these biological processes (Additional file 6: Figure S3)..
- Particularly, PvRbohB silencing showed a marginal reduction in the growth of the main root and a significant reduction in lateral root density [13].
- In this work, the ex- pression levels of the cell wall-remodeling genes, as well as genes involved in the cell cycle and the biosynthesis of and response to auxins, were considerably downregulated in the PvRbohB-silenced roots (Fig.
- Under symbiotic conditions, PvRbohB silencing affected the expression of approximately half the number of DEGs in the nonsilenced roots under nodulation and mycorrhiza- tion conditions.
- The last of these proteins is one of the most important enzymes involved in the regulation of glycoprotein oligosaccharide biosynthesis.
- The ef- fect of PvRbohB silencing on the transcriptional re- sponse of this battery of genes supports the defects observed in the nodulation program of the RbohB- RNAi roots [13].
- Several Rboh members are known to be important players in the developmental programs of different tissues and organs in a variety of plant species .
- Here, we also observed the induction of the early auxin response genes, such as SAUR32/71, GH3.5, and IAA28, in the rhizobia-inoculated roots.
- Approximately 45% of the auxin- related genes observed in the rhizobia-inoculated control roots presented different expression patterns in the PvRbohB-RNAi roots, including SAUR32, IAA13, and DRMH1 (Fig.
- Similar to our observations in the rhizobia- inoculated roots, the early auxin response genes, such as IAA17, GH3.6, and SAUR32/71, were induced in the mycorrhized control roots, while ARF7 was repressed (Fig.
- PvRbohB-RNAi abol- ished the differential expression levels of these genes in both the root nodule and AM symbioses, although genes such as GH3.1 were induced in the rhizobia-inoculated roots (Fig.
- These data suggest that PvRbohB has an important function in the regulation and response to auxin in both microbial symbioses..
- In this study, PvCKX2 and PvCKX5 were induced in the rhizobia-inoculated control roots, confirming the role of the cytokinins at the early stages of nodulation in P..
- These genes were downregulated in the PvRbohB-silenced roots, however, in which only CKX6 and GLUCOSYL/GLUCURONOSYL TRANSFER- ASE were upregulated, suggesting a crucial role for PvRbohB in regulating cytokinin biosynthesis during nodulation in P.
- Our data generally support this conclusion, but also show that PvRbohB plays a role in the biosynthesis of cy- tokinins during P.
- This nodulation-related mi- tosis was affected in the PvRbohB-RNAi transgenic roots, which showed a drastic reduction in mitotic activ- ity compared to the infected control roots [13], which are not required for mycorrhizal colonization..
- In this work, genes involved in the biosynthesis and response of ethylene presented differential expression in control and PvRbohA-RNAi roots (Fig.
- Particularly, the homologs of two key ethylene biosynthesis regulators, 1-AMINOCYCLOPRO- PANE-1-CARBOXYLATE (ACC) SYNTHASE and ACC OXIDASE, were upregulated in the control roots, con- firming previous reports of an increase in ethylene pro- duction during the early stages of nodulation in P..
- Our results confirm the central role of auxins, cy- tokinins, and ethylene in the nodulation and mycorrhization processes in P.
- vulgaris and suggest that PvRbohB could be an important player in the homeosta- sis of these phytohormones in both microbial symbioses..
- These results provide important information on the symbiotic gene signaling networks involved in the early stages of rhizobial and mycorrhizal colonization as well as the differential effects of a NADPH oxidase gene, RbohB, on these processes..
- Also, the Pearson’s cor- relation coefficients between the replicates were estimated using the cor function of the ggpubr package and plotted with the function ggplot of the ggplot2 package all in R to confirm the clustering observed in the MDS plots.
- The distribution and abundance (Log2Fold- Change) of these genes were presented in Venn dia- grams using the function draw.quad.venn in the VennDiagram package in R and in heatmaps with the function heatmap.2 of the gplot package in R, respect- ively.
- Functional annotation of the DEGs in rhizobia-inoculated (Rhiz) and mycorrhized (Myc) roots of P.
- Functional annotation of the DEGs in the PvRbohB-RNAi roots in nonsymbiotic conditions of P.
- Functional annotation of the DEGs in rhizobia-inoculated and mycorrhized PvRbohB-RNAi roots, relative to the control.
- Transcript levels of PvRbohB in the control and PvRbohB-RNAi roots used in this study.
- UNAM-DGAPA and CONACyT-FC were not involved in the design of the study, collection, analysis, and interpretation of data and in writing the manuscript..
- Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth.
- Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance.
- Transcriptional regulation of ROS controls transition from proliferation to differentiation in the root.
- Hydrogen peroxide-regulated genes in the Medicago truncatula – Sinorhizobium meliloti symbiosis.
- Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus Fungi provide insights into the genetic program activated during Arbuscular Mycorrhiza

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