https://orcid.org/0000-0001-6224-675X
Figures
Abstract
Plant phospholipase C (PLC) proteins are phospholipid-degrading enzymes classified into two subfamilies: phosphoinositide-specific PLCs (PI-PLCs) and non-specific PLCs (NPCs). PI-PLCs have been widely studied in various biological contexts, including responses to abiotic and biotic stresses and plant development; NPCs have been less thoroughly studied. No PLC subfamily has been characterized in relation to the symbiotic interaction between Fabaceae (legume) species and the nitrogen-fixing bacteria called rhizobia. However, lipids are reported to be crucial to this interaction, and PLCs may therefore contribute to regulating legume–rhizobia symbiosis. In this work, we functionally characterized NPC4 from common bean (Phaseolus vulgaris L.) during rhizobial symbiosis, findings evidence that NPC4 plays an important role in bean root development. The knockdown of PvNPC4 by RNA interference (RNAi) resulted in fewer and shorter primary roots and fewer lateral roots than were seen in control plants. Importantly, this phenotype seems to be related to altered auxin signaling. In the bean–rhizobia symbiosis, PvNPC4 transcript abundance increased 3 days after inoculation with Rhizobium tropici. Moreover, the number of infection threads and nodules, as well as the transcript abundance of PvEnod40, a regulatory gene of early stages of symbiosis, decreased in PvNPC4-RNAi roots. Additionally, transcript abundance of genes involved in autoregulation of nodulation (AON) was altered by PvNPC4 silencing. These results indicate that PvNPC4 is a key regulator of root and nodule development, underscoring the participation of PLC in rhizobial symbiosis.
Citation: Pacheco R, Juárez-Verdayes M, Chávez-Martínez AI, Palacios-Martínez J, Leija A, Nava N, et al. (2025) The non-specific phospholipase C of common bean PvNPC4 modulates roots and nodule development. PLoS One 20(5): e0306505. https://doi.org/10.1371/journal.pone.0306505
Editor: Ying Ma, Universidade de Coimbra, PORTUGAL
Received: June 18, 2024; Accepted: December 20, 2024; Published: May 5, 2025
Copyright: © 2025 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files
Funding: This research was partially funded by the National Council of Humanities, Science and Technology of Mexico (CONAHCyT) under Frontiers of Science (FC) CF-2023-I-297 to C. Quinto. CONAHCyT also funded a PhD fellowship to R. Pacheco (749422), a postdoctoral fellowship to J. Palacios Martínez (2342655), and a postdoctoral fellowship to A. I. Chávez Martínez.” The Program to Support Research and Technological Innovation Projects/UNAM (PAPIIT) awarded grants N203021 and IN204024 to C. Quinto and IN204024 to R. Pacheco. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Rhizobial symbiosis is established between legume species (Fabaceae) and nitrogen-fixing soil bacteria called rhizobia. This interaction normally occurs in nitrogen-poor soils, when roots exude flavonoids into the rhizosphere that are specifically recognized by rhizobia. The specific recognition of flavonoids induces the synthesis and secretion by rhizobia of lipochitooligosaccharides known as nodulation factors (NFs), which are perceived by receptors at the plasma membrane of root hairs [1,2]. The perception of NF triggers the so-called common symbiotic pathway, which allows rhizobial infection and the initiation of nodule organogenesis [3]. The most common route of rhizobia infection occurs through the formation of a tubular structure known as the infection thread (IT) at the apex of root hairs. Bacteria migrate through the IT to the growing nodule and differentiate into bacteroids, giving rise to nitrogen-fixing organelles called symbiosomes. In the nodules, the nitrogen fixed (in the form of ammonia) by the bacteroids is exchanged for the carbon produced by the plants, providing a source of energy for the bacteria [4].
The establishment of rhizobial symbiosis involves several biological processes, such as: hormone-mediated signaling [5], reactive oxygen species signaling [6,7], autoregulation of nodulation [8–10], membrane turnover, and lipid-mediated signaling and metabolism [11,12]. The involvement of lipids in the establishment of this interaction has been reported to be crucial on the basis of pharmacological studies [13–15]. For example, phospholipids are thought to play a role in the NF-induced signal transduction pathway [15]. Moreover, lipid turnover in membranes has been demonstrated to be essential for nodulation [11]. In this scenario, phospholipases appear to be key regulators of symbiosis, as they hydrolyze phospholipids, the main components of membranes. Plant phospholipases are divided into three classes: A, C, and D. Specifically, phospholipases C (PLCs) cleave the glycerophosphate bond, releasing diacylglycerol and the phosphorylated head group [16]. Based on their substrate specificity, PLC members are classified into two groups or subfamilies: 1) phosphoinositide-specific PLCs (PI-PLCs) and 2) non-specific PLCs (NPCs) that non-specifically hydrolyze major membrane phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [17].
The structure of PI-PLC is characterized by the presence of two catalytic domains, PLC-X and PLC-Y; an EF-hand calcium-binding motif at their N terminus; and a C2 calcium-binding domain at their C terminus [18]. However, the EF-hand motif has not been reported in all PI-PLCs [19,20]. Unlike PI-PLCs, NPCs have a single catalytic domain, the phosphoesterase domain, and no EF-hand motif [19–21]. The functions of NPCs have been studied in abiotic stress [22], in plant immunity [23], and during plant development, specifically root development [24,25]. Their participation in rhizobial symbiosis remains unexplored to date. In fact, neither PI-PLC nor NPC from common bean (Phaseolus vulgaris L.) have been functionally characterized. In this work, we performed an in silico analysis of the PLCs in common bean (PvPLCs) and functionally characterized the PvNPC4 gene during bean root development and in the symbiosis with Rhizobium tropici.
Materials and methods
In silico analysis of phospholipase C (PLC) family members
To identify members of the PLC family in common bean (Phaseolus vulgaris L.) and barrelclover (Medicago truncatula Gaertn.), a search was performed on the corresponding genome sequences (v2.1-common bean and Mt4.0v1, respectively) available at the Phytozome 13 database [26] (https://phytozome.jgi.doe.gov/pz/portal.html) using the BLASTP algorithm. Lotus japonicus (Regel) K. Larsen, PLCs were identified in Lotus Base (https://lotus.au.dk/blast/#) MG20 v3.0. For BLASTP, the PLC protein sequences previously reported in Arabidopsis (Arabidopsis thaliana (L.), Heynh. were used as queries [27,28]. Additionally, the previously reported soybean (Glycine max) (L.) Merr. PLC protein family was searched and any missed proteins from the published list were added [21,22]. Conserved domains in putative PLC members were identified using SMART (http://smart.embl-heidelberg.de/) [29]. For phylogenetic analysis, the PLC protein sequences of Arabidopsis, soybean, and common bean were used. Sequences were aligned using the MUSCLE algorithm and manually edited using MEGA v. X [30] to remove misaligned sequences. The phylogenetic tree was reconstructed using the IQ-TREE algorithm version 1.6.12 [31] and the maximum-likelihood method based on JTT + G model with 10,000 bootstrap replicates.
Physicochemical parameters were predicted using ExPASy (https://web.expasy.org/cgi-bin/protparam/protparam) [32]. The subcellular localization of PvPLCs was predicted using WOLF-PSORT (https://wolfpsort.hgc.jp/), CELLO ver.2.5 (http://cello.life.nctu.edu.tw/) [33], and LocTree3 (https://rostlab.org/services/loctree3/) [34]. MEME ver. 5.4.1 (https://meme-suite.org/meme/tools/meme) [35] was used to identify conserved motifs from PvPLCs with the maximum number of motifs of 10, and the other parameters were set to default. The gene structure was analyzed by TB tools software [36].
The chromosomal position of PvPLCs was determined according to the Phytozome v13 database, and the chromosome distribution was visualized using TB tools software. The One-Step MCScanX-SuperFast program of TB tools software was used to perform pairwise duplication and synteny analysis. The cis-elements in the promoter regions (2000 bp upstream of the translation start codon) were analyzed using the PlantCARE online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and plotted using TB tools software. The nonsynonymous (Ka) and synonymous (Ks) substitution rates of each gene pair were calculated using TB tools software.
The expression profiles of PvPLC genes in various plant tissues at different developmental stages were retrieved from the P. vulgaris gene expression atlas database PvGEA (https://www.zhaolab.org/PvGEA/) [37]. Data were downloaded as reads per transcript kilobase per million reads mapped (RPKM). Heatmaps representing the expression profile of PvPLCs were plotted using TB tools software.
Growth conditions of wild-type plants
Seeds of wild-type P. vulgaris L. cv. Negro Jamapa plants were germinated according to the protocol published at dx.doi.org/10.17504/protocols.io.261ge3bpjl47/v3 [38].
Two-day-old seedlings were planted in pots containing autoclaved vermiculite and watered every 3 days with sterile deionized water for 1 week. After 1 week, seedlings were grown in pots containing autoclaved vermiculite and inoculated with Rhizobium tropici CIAT 899 [39] at an OD600 of 0.05 or not inoculated as control. Control plants were watered every 3 days with a solution of B & D medium [40] containing 2 mM KNO3 and 0.015 mM (NH4)2SO4 to prevent rhizobial infection. Similarly, inoculated plants were watered every 3 days with nitrate-free B & D solution [40].
Roots samples were collected at 12, 24, and 72 h post inoculation (hpi) and at 14 days post inoculation (dpi). All tissues were stored at −75°C until analysis. The roots of 14 dpi plants were free of nodules. Additionally, leaves, stems, and roots were collected from seedlings irrigated with deionized water, at 7 days after transplanting into pots, and root hairs from 2-day-old seedlings. These tissues were used for total RNA extraction and quantification of Pv-PI-PLC4 or PvNPC4 transcript abundance by reverse-transcription quantitative PCR (RT-qPCR), as described below. The transcript abundance of PvNPC4 in seedling tissues 7 days after transplanting to pots, was compared with the transcriptional profile of PvNPC4 in different tissues and stages obtained from the transcriptional landscape https://www.zhaolab.org/PvGEA/ [37].
RNA extraction and RT-qPCR assays
Total RNA extraction and complementary DNA synthesis were performed following the protocol published at dx.doi.org/10.17504/protocols.io.8epv5jq24l1b/v1. qPCR assays were conducted with a Maxima SYBR Green/ROX qPCR Master Mix (2X) kit (Thermo Scientific, Waltham, MA, USA) on a qPCR system (QuantStudio 5; Applied Biosystems, Waltham, MA, USA). The qPCR steps consisted of 95°C for 10 min, 30 cycles of 95°C for 15 s, and 60°C for 60 s. Relative transcript abundance were calculated with the 2−ΔΔCT method [41] using elongation factor 1α (EF1α, Phvul.004G075100.1) and β-tubulin (Phvul.009G017300.1) as reference genes. The primer sequences used in all qPCR assays are given in S1 Table.
Plasmid construction
For knockdown of PvNPC4, an RNAi silencing construct (PvNPC4-RNAi) was generated. To generate the PvNPC4-RNAi construct, a specific 131-bp fragment from the PvNPC4 coding sequence was amplified using a common bean root cDNA as a template. This amplicon was inserted into the pENTRTM/D-TOPOTM vector generating the donor vector. The donor vector was recombined with the binary vector pTDT-DC-RNAi [42] using GATEWAYTM technology to yield the PvNPC4 silencing construct (S1 Fig). The correct orientation of the inserted fragment in the donor and binary vector was verified by PCR and Sanger sequencing using the appropriate primers (S1 Table). The pTdT-SAC vector carrying a truncated and unrelated sequence from Arabidopsis pre-MIR159 was used as control [43].
Generation of composite plants and selection of Agrobacterium rhizogenes clones of interest
Transgenic hairy roots were generated in wild-type plants of Negro Jamapa (composite plants) following the protocol described at dx.doi.org/10.17504/protocols.io.261ge3bpjl47/v3 [38]. Selection of A. rhizogenes clones for PvNPC4-RNAi knockdown was performed according to the criteria discussed previously [38]. To generate hairy roots, one A. rhizogenes clone transformed with the control vector was used, as well as several A. rhizogenes clones transformed with the PvNPC4 silencing construct. The roots of common bean plants were inoculated with R. tropici CIAT899 or watered with B & D medium only and harvested at 10 dpi for RNA extraction, as described previously dx.doi.org/10.17504/protocols.io.8epv5jq24l1b/v1. The silencing efficiency of PvNPC4 by the RNAi construct was confirmed by RT-qPCR analysis; two A. rhizogenes clones transformed with the RNAi construct able to reduce PvNPC4 transcript abundance in more than 60%, were selected (S2 Fig).
Phenotypic analysis
The phenotypic analysis was performed on roots inoculated or not with R. tropici CIAT899 or R. tropici transformed with the β-glucuronidase gene (R. tropici-GUS). To examine the effect of PvNPC4 silencing on root development, the length of primary roots was measured, and the number of primary and lateral roots were counted. To examine the effect of PvNPC4 silencing on rhizobial symbiosis, the number of ITs was quantified in hairy roots at 10 dpi with R. tropici-GUS, as well as the number of nodules at 14 dpi with R. tropici CIAT899.
Analysis of gene expression in transgenic roots
To search for the role of PvNPC4 in regulating root development, we analyzed the transcript abundance of the auxin transporter gene Pin-formed 1 (PvPin1b, Phvul.004G150600.1) [44] and two genes, LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16)/ASYMMETRIC LEAVES2-LIKE 18 (ASL18) (PvASL18a, Phvul.001G159300.1 and PvASL18b, Phvul.001G159300.1) by RT-qPCR. PvASL18a and PvASL18b were identified by BLAST-P in the Phytozome 13 [26] database using the LOB Domain IPR004883 sequence from AT2G42430.1. RT-qPCR analysis was performed using mRNA from hairy roots of uninoculated 10-day-old seedlings, carrying the PvNPC4-RNAi construct and the control plasmid. Hairy roots were collected from inoculated seedlings at 10 dpi for the quantification of genes related to rhizobial symbiosis: Nodule Inception (PvNIN, Phvul.009G115800.1), Early nodulin 40 (Enod40, Phvul.002G064166.1[45]), To Much Love (PvTML1a, Phvul.001G094400.1), and Rhizobia-Induced CLE1 and 2 (PvRIC1, Phvul.005G096901.1; PvRIC2, Phvul.011G135900.1 [10]). PvTML1a was identified in the common bean genome available in Phytozome 13 [26] using the Kelch repeat domain sequence of F-box IPR052439 of AT3G27150.1 and AT5G40680.1. Primers for qPCR assays are listed in S1 Table.
Statistical analysis
For statistical analysis, a bootstrap test with 9999 samplings with replacement and a Monte Carlo simulation test with 9999 samplings without replacement were used. These analyses require fewer assumptions than traditional methods and are more accurate than classical statistical analyses [46,47]. These methods make it possible to compare the value of a given statistic with a reference distribution generated from the data itself. Importantly, the Monte Carlo simulation is useful to estimate the p-value when the sample size is small [48], which was commonly the case in this study. Both tests were performed in RStudio 4.1.2 and 4.3.1 (http://www.rstudio.com/) using the tidyverse package [49]. RAWGraphs (https://app.rawgraphs.io/) was used to identify outliers in all datasets. Spearman correlation between PvNPC4 transcript abundance in common bean tissues and its expression profile available in PvGEA obtained by RNA-seq [50] was performed in RStudio 4.3.
Results
Identification of PLCs in legume species
To evaluate the putative role of PLCs in the symbiotic relationship between legumes and rhizobia, we asked whether PLC genes constitute a gene family in the genomes of several model legumes, such as barrelclover, L. japonicus, and common bean. To this end, we queried the corresponding legume genome databases using Arabidopsis protein sequences as queries. After discarding redundant sequences and confirming conserved domains using SMART, we identified 15 non-redundant PLCs in barrelclover and 12 PLCs each in L. japonicus (S2 Table) and common bean (S3 Table). The same analysis returned 13 PI-PLC members, instead of the published number of 12, and 9 NPC members rather than the published number of 7 in the soybean genome [21,22]; we provide the updated list of GmPLCs in S2 Table.
In common bean, the amino acid sequences of seven PvPLCs showed the characteristic domains of the PI-PLC subfamily, while the remaining five PvPLCs displayed the phosphoesterase domain associated with members of the NPC subfamily (S3 Fig). Moreover, all putative PvPI-PLCs above presented the catalytic PLC_X and PLC_Y domains, as well as the Ca2+ and phospholipid-binding domains; we identified the EF-hand motif in three of the seven PvPI-PLCs. Furthermore, only PvPI-PLC5 had a predicted signal peptide (S3 Fig). For the predicted PvNPCs, all had the characteristic phosphoesterase domain, and only PvNPC4 lacked a predicted signal peptide (S3 Fig). We named all PLCs based on their subfamily classification and chromosomal location (S3 Table).
PLCs of legume species are phylogenetically close
To analyze the function of PvPLCs in the symbiotic interaction between legumes and rhizobia, we reconstructed a phylogenetic tree using the amino acid sequences of all model legume species used for the identification of PLC members. We also included non-legumes as reference: Arabidopsis, a non-legume dicotyledonous species, and rice (Oryza sativa L.), a monocotyledonous species. Phylogenetic reconstruction separated all these PLCs into two well-defined clades, corresponding to PI-PLCs and NPCs (Fig 1). Importantly, NPCs clustered closer to the outlier sequence used here, a Mycobacterium tuberculosis PLC sequence, than to the PI-PLC clade. Within the PI-PLC and NPC clades, PLCs from non-legume species (Arabidopsis and rice) were the most phylogenetically distant from those from legume species. Importantly, PLCs from legume species were more closely related to each other than to non-legumes, with the smallest phylogenetic distance being between common bean and soybean (Fig 1 and S4 Fig). Together, these results suggest an ancient evolutionary divergence between legume and non-legume PLCs.
The plant species used in this analysis are M. truncatula (Medtr), L. japonicus (Lj), G. max (Glyma), P. vulgaris (Phvul), A. thaliana (AT), and O. sativa (LOC Os). Blue indicates members from the PI-PLC clade; green indicates members from the NPC clade. The amino acid sequence of M. tuberculosis PLC (YP_009359121.1) was used as an outlier. The phylogenetic tree was reconstructed using the IQ-TREE algorithm based on the maximum-likelihood method and the JTT + G model; 10,000 bootstraps were performed.
To better typify PvPLCs, we analyzed some of their physicochemical properties (S4 Table). PvPI-PLC members were longer than PvNPCs, ranging from 550 to 636 amino acids compared to 487–525 amino acids for PvNPCs and therefore had higher molecular weights. Furthermore, PvPI-PLCs showed a heterogeneous isoelectric point (pI): four had a slightly acidic pI, one had a neutral pI, and two had a slightly alkaline pI. In contrast, four of the five PvNPCs showed a slightly acidic pI; only PvNPC1 had a marginally alkaline pI. The grand average of hydropathy (GRAVY) values for all PvPLCs were predicted to be less than 0, indicating their hydrophilic behavior.
The PvPLC subfamily members presented different exon-intron structures; their encoded proteins also displayed distinct protein motifs. We detected nine exons in PvPI-PLC genes and three or four exons in PvNPC genes (S5 A, B Fig). We identified no 3′ untranslated region (UTR) in PvPI-PLC7 and neither 5′ UTR nor 3′ UTR in PvNPC4 and PvNPC5 (S5A, B Fig). In the PvPLC protein sequences, we identified 10 protein motifs; PvPI-PLCs contain motifs 1–8, while PvNPC members contain motifs 4, 5, 7, 9, and 10 (S5C Fig).
PvPLC genes have undergone duplication
We assigned all PvPLC genes to chromosomes in the common bean genome, as described in Materials and methods. Eleven of the PvPLC genes were located on 5 of the 11 chromosomes of common bean as follows: three genes on chromosome 1, two genes on chromosome 6, four genes on chromosome 8, one gene on chromosome 9, and one gene on chromosome 10. The remaining gene currently maps to a scaffold (S6 Fig). The three genes on chromosome 1 and the four genes on chromosome 8 correspond to PI-PLC genes. The three genes on chromosome 1, as well as three of the four genes on chromosome 8, appear in tandem along the chromosome (S6 Fig). In contrast, NPC genes are widely distributed along the three chromosomes to which they map, with no evidence of tandem arrangement (S6 Fig).
To explore the evolutionary relationships between the PvPLC genes in common bean, we performed a synteny analysis using the program MCScanX. The analysis suggested two segmental duplication events corresponding to the PvPLC1–PvPLC4 and PvPLC1–PvPLC7 gene pairs and one tandem duplication event for the PvPLC4–PvPLC7 gene pair (Fig 2A). We detected no collinearity among the PvNPC genes. When we calculated the ratios of nonsynonymous to synonymous changes (Ka/Ks) between PvPLC pairs, we obtained values below 1 (S3 File), suggesting that they underwent purification and selection after duplication. A synteny analysis between common bean and soybean identified 16 orthologous pairs, emphasizing the close evolutionary relationship between these two legume species (Fig 2B).
(A) PvPLC gene duplication analysis. (B) Collinearity analysis of PLC genes in common bean and soybean. Black lines show collinearity between PvPLC genes; red lines show collinearity between PvPLC and GmPLC genes.
Identification of cis-regulatory elements in the promoter region of PvPLC genes
To better understand the regulation of PvPLC gene expression, we looked for cis-elements located in their promoter regions (up to 2000 bp upstream of the translation start site) using the online tool PlantCARE. We identified 57 cis-acting elements clustered into three groups: light responsive, phytohormone responsive, and stress responsive (S3 File). The cis-acting elements in the PvNPC4 promoter were classified as follows: seven related to light and stress sensitivity, three to growth and developmental sensitivity, and eight to sensitivity to phytohormones (Table 1). Among the elements related to light, box-4 and G-box were the most represented; the main stress-related elements represented were anoxic responsive (ARE) and W-box. Among growth and development cis-elements, the CAT-box was present in the greatest number of copies, with TCA-element being the most frequent cis-element related to phytohormones (Table 1).
Expression of PvPI-PLC4 and PvNPC4 genes varies depending on plant tissue and development and in response to rhizobia inoculation
We analyzed the expression patterns of genes belonging to the PvPLC family in different common bean tissues. Since the PvPLC family comprises several genes, we selected two genes, PvPI-PLC4 and PvNPC4, for characterization in bean–rhizobia symbiosis. This selection was based on their expression profiles reported in the common bean gene expression atlas (PvGEA) as well as their phylogenetic relationship with Arabidopsis PLC genes. PvPI-PLC4 showed the highest transcript abundance (in RPKM) in roots and nodules (S7 Fig). PvNPC4 is phylogenetically closer to Arabidopsis NPC4 (At3g03530) (S4 Fig) which is reported to be involved in the degradation of plasma membrane phospholipids [28]. Considering that phospholipid metabolism is crucial for nodule organogenesis [11,12], it was of interest to select this gene to study its role in rhizobial symbiosis. To examine the expression profile of these two common bean genes during symbiosis, we quantified their transcript abundance at different stages of symbiosis with R. tropici using RT-qPCR analysis.
Unexpectedly, we barely detected PvPI-PLC4 transcript abundance in roots and nodules of common bean under our conditions. We observed no significant differences between inoculated and non-inoculated roots at 12, 24, or 72 h post inoculation (hpi), with p-values of 0.25, 0.86, and 0.7, respectively (S8A Fig). Likewise, PvPI-PLC4 transcript was not significantly different at 14 dpi between inoculated roots and nodules compared to non-inoculated control roots, with p-values of 0.6 and 0.9, respectively. Moreover, no significant difference was observed in inoculated roots compared to nodules, with a p-value of 0.6 (S8B Fig).
Relative PvNPC4 transcript abundance during rhizobial symbiosis did not change significantly in inoculated roots, at 12 or 24 hpi compared to non-inoculated roots (p = 0.28 and 0.354, respectively). However, at 72 hpi, relative PvNPC4 transcript abundance significantly increased in inoculated roots compared to non-inoculated roots (p = 0.02) (Fig 3A). In contrast, relative PvNPC4 transcript abundance significantly decreased in inoculated roots (p = 0.024) and nodules (p = 0.029) compared to non-inoculated roots at 14 dpi (Fig 3B). To further investigate the changes in PvNPC4 transcript abundance upon rhizobium inoculation, we analyzed PvNPC4 transcript abundance in inoculated and non-inoculated roots over time (12, 24, and 72 hpi, and 14 days post inoculation [dpi]).
(A) Relative abundance of PvNPC4 transcript at early stages after inoculation with R. tropici. (B) Relative abundance PvNPC4 of transcript at 14 dpi with R. tropici. Roots-, not inoculated roots; Roots + , inoculated roots without nodules. The lower and upper edges of boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. Statistical significance was assessed with a Monte Carlo simulation test with 9999 resamples without replacement (* p ≤ 0.05). Black dots in the box plots indicate independent samples from three biological replicates.
In non-inoculated roots, PvNPC4 transcripts showed two points of high abundance, at 12 h and 14 days, while decreasing between 24 and 72 h (S9A Fig). These two high points of transcript abundance were significantly different compared to the transcript abundance at 72 h (p < 0.001). In inoculated roots, we observed three high points of PvNPC4 transcript abundance, at 24 hpi and 72 h, and decreased considerably in roots and nodules at 14 dpi (S9B Fig). Statistical analysis showed differences in inoculated roots of 12 dpi compared to roots of 14 dpi (p = 0.04) and nodules of 14 dpi (p = 0.03). Moreover, significant differences were observed in roots of 24 hpi compared to roots of 14 dpi (p = 0.049) and nodules of 14 dpi (p < 0.001).
Additionally, we quantified PvNPC4 transcript abundance in leaves, stems, and roots of seedlings of 7 days after transplanting into pots irrigated with deionized water, and in root hairs of 2-day-old seedlings. We observed the lowest expression of PvNPC4 in leaves and the highest in roots compared to the transcriptional landscape obtained from PvGEA (S10A Fig). Importantly, a Spearman correlation indicated that the transcript abundance obtained by qPCR and the PvGEA transcriptional profile obtained by RNA-seq are not significantly different (S10C Fig). Additionally, the transcriptional landscape of AtNPC4, the ortholog of PvNPC4, obtained from Arabidopsis thaliana Single Cell Atlas Data also showed the highest expression in roots (S10B Fig). Together, these findings suggest that PvNPC4 may play an important role in roots by modulating its expression in response to rhizobial infection.
Knockdown of PvNPC4 negatively affects root development and nodule formation
To explore the involvement of PvNPC4 during rhizobial symbiosis, we examined the nodulation phenotype in hairy roots carrying a PvNPC4-RNAi construct for PvNPC4 knockdown, using hairy roots carrying the empty vector as a control. To this end, we used two independent A. rhizogenes clones carrying the PvNPC4-RNAi vector, namely, RNAi-C1 and RNAi-C8, whose silencing efficiency was previously validated at 10 dpi with R. tropici (S2 Fig). Surprisingly, PvNPC4-RNAi seedlings developed much shorter and fewer roots compared to the control vector (Fig 4D). Indeed, the length of primary PvNPC4-RNAi roots was about half that of roots carrying the control vector, a difference that was significant (p < 0.001) (Fig 4A). Similarly, PvNPC4-RNAi roots had fewer primary and lateral roots compared to the control (Fig 4B, C).
(A) Length (cm) of primary hairy roots, (B) number of primary hairy roots, and (C) number of lateral hairy roots, (D) transgenic roots carrying the RNAi control vector or the PvNPC4 silencing construct. Ctrl-RNAi indicates roots carrying the control vector. PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. For statistical analysis, a bootstrap test was performed with 9999 samples with replacement (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Black dots in the box plots indicate individual samples from four biological replicates.
Auxin is a key regulator of root development [51], which prompted us to explore the relative transcript abundance of genes involved in the auxin-mediated signaling in 10-day-old transgenic hairy roots irrigated with B & D medium. Among the genes related to auxin signaling, LBD16/ASL18, has been shown to participate in root development [52]. Through in silico analysis, two LBD16/ASL18 orthologs were identified in common bean, PvASL18a and PvASL18b, whose expression profiles in the PvGEA and in an Open Big Data metatranscriptome showed a high expression level in roots and nodules of common bean (S5 File). Regarding auxin transport, the common bean genome encodes 16 genes involved in this process [44], showing that the auxin transporter PvPin1b, is the most abundant transcript in roots and nodules according to the profile in PvGEA (S5 File).
PvASL18a and PvASL18b transcript abundance was slightly and significantly (p < 0.001) reduced, respectively, in PvNPC4-RNAi roots compared to control (Fig 5A). On the other hand, the abundance of PvPin1b transcripts decreased significantly (p = 0.02) by approximately 50% in PvNPC4-RNAi hairy roots compared to control roots (Fig 5B). These findings strongly suggest that PvNPC4 in common bean is involved in root development in an auxin-dependent manner. This finding deserves further investigation; however, it is outside our focus regarding the role of the PvNPC4 gene in the mutualistic interaction between common bean roots and rhizobia.
Ctrl-RNAi indicates transgenic roots carrying the control vector (pTdT-SAC). PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of the boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the maximum and minimum values in the data set. For statistical analysis, a Monte Carlo simulation test was used with 9999 samples without replacement (* p ≤ 0.05, **** p ≤ 0.0001). Black dots in the box plots indicate individual samples from three biological replicates.
PvNPC4 transcript abundance differentially changed in rhizobium-inoculated roots and in nodules at the early and mature stages of symbiosis (Fig 3). This observation prompted us to analyze the participation of PvNPC4 in nodule development. To this end, we quantified the number of ITs and the number of nodules at 10 and 14 dpi with R. tropici. Importantly, the number of ITs in PvNPC4-RNAi hairy roots at 10 dpi was significantly lower than that in the control (Fig 6A) (p < 0.001). In agreement with this result, the number of nodules at 14 dpi also decreased following PvNPC4 silencing (p = 0.0067) (Fig 6B).
(A) Number of infection threads (ITs) per root at 10 dpi. (B) Number of nodules per root at 14 dpi. Ctrl-RNAi indicates transgenic roots carrying the control vector pTdT-SAC. PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of the boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. For statistical analysis of IT numbers, a Monte Carlo simulation test was used with 9999 samples without replacement (**** p ≤ 0.0001; two biological replicates). For the number of nodules, a bootstrap test was performed with 9999 samples with replacement (* p ≤ 0.01; three biological replicates). Black dots in the box plots indicate individual samples.
To better understand the regulatory function of PvNPC4 in rhizobial symbiosis, we quantified the transcript abundance of the two early nodulin genes PvNIN and PvEnod40 in transgenic roots inoculated with R. tropici (10 dpi), as markers of symbiosis progression. We did not detect statistical differences in the transcript abundance of PvNIN in PvNPC4-RNAi roots compared to control roots (Fig 7A). However, PvEnod40 transcript abundance was significantly decreased (p < 0.001) in PvNPC4-RNAi roots at 10 dpi (Fig 7B). Overall, these findings suggest that PvNPC4 participates in rhizobial symbiosis in common bean by modulating the expression of some early nodulins.
Relative transcript abundance of PvNIN (A), PvEnod40 (B), and PvRbohA (C) at 10 dpi. Ctrl-RNAi indicates transgenic roots carrying the control vector pTdT-SAC. PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. For statistical analysis, a Monte Carlo simulation test was used with 9999 samples without replacement (**** p ≤ 0.0001. Black dots in the box plots indicate individual samples from three biological replicates.
Previous studies by our group have shown the important role of NADPH oxidases, known in plants as RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs), in rhizobial symbiosis [6,7]. Moreover, PLCs and RBOHs have been shown to participate in plant response mechanisms in biotic interactions [23,53]. Therefore, we analyzed the transcript abundance of PvRbohA, a gene encoding a NADPH oxidase, in inoculated and non-inoculated PvNPC4-RNAi hairy roots at 10 dpi. No significant differences were observed between PvNPC4-RNAi roots and RNAi control roots in inoculated or non-inoculated roots (Fig 7C). In both inoculated and non-inoculated roots, PvRbohA transcript abundance was slightly increased in PvNPC4-RNAi roots compared to RNAi control roots. Importantly, PvRbohA transcript abundance was very similar between inoculated and non-inoculated PvNPC4-RNAi roots (Fig 7C), indicating that PvNPC4 silencing can affect PvRbohA expression regardless of rhizobial infection.
To examine the possible role of PvNPC4 in the autoregulation of nodulation (AON) pathway [54], transcript abundance of AON-related genes, such as RIC and TML, was quantified in control and PvNPC4-RNAi roots, non-inoculated or inoculated with R. tropici (10 dpi). It was previously reported that P. vulgaris RIC1 and RIC2 were induced by R. phaseoli in common white bean [10]. Along these same lines, we quantified the abundance of PvRIC1 and PvRIC2 transcripts in PvNPC4-RNAi and RNAi control roots, non-inoculated or inoculated with R. tropici (10 dpi). In non-inoculated roots, no statistical difference in PvRIC1 transcript abundance was observed between control and PvNPC4-RNAi roots (Fig 8A). However, in inoculated roots, the transcript abundance of this gene was significantly induced (p < 0.001) in PvNPC4-RNAi roots compared to RNAi control roots (Fig 8A). Additionally, PvRIC1 transcript abundance in inoculated PvNPC4-RNAi roots was higher (p < 0.001) than that in non-inoculated PvNPC4-RNAi roots (Fig. 8A) indicating that PvNPC4 and PvRIC1 are functionally interconnected during rhizobial symbiosis.
Relative transcript abundance of PvRIC1 (A), PvRIC2 (B), and PvTLMa (C). Ctrl-RNAi indicates transgenic roots carrying the control vector pTdT-SAC. PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of the boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. For statistical analysis, a Monte Carlo simulation test was used with 9999 samples without replacement (**** p ≤ 0.0001). Black dots in the box plots indicate individual samples from three biological replicates.
Regarding transcript abundance of PvRIC2, no statistical differences were found between PvNPC4-RNAi roots and RNAi control roots either in non-inoculated or inoculated roots (Fig 8B). Interestingly, PvRIC2 transcript abundance in inoculated PvNPC4-RNAi roots was significantly reduced compared to that in non-inoculated PvNPC4-RNAi roots (Fig 8B), likely as a compensatory mechanism for RIC expression in this specific stage of common bean-R. tropici symbiosis. Transcription profiles of common bean TML genes were first examined using data from PvGEA and raw data available in Open Big Data. Through this analysis, PvTMLa was identified as having the highest transcript abundance in roots and nodules (S4 File). Using qPCR analysis, we detected a significant reduction in PvTMLa transcript abundance (p < 0.001) in PvNPC4-RNAi roots compared to control roots (Fig 8C). In contrast, in inoculated roots no differences were observed between PvNPC4-RNAi roots and control roots (Fig 8A).
The ASL18/LBD16 genes have been shown to be involved in root and nodule development [52,55], therefore, we examined the effect of R. tropici on the transcript abundance of these genes. For this, transcript abundance of PvASL18a and PvASL18b was quantified in PvNPC4-RNAi and control roots at 10 dpi and compared to non-inoculated roots. As observed previously (Fig 5A), in non-inoculated roots, PvASL18a transcript abundance did not change in PvNPC4-RNAi roots compared to RNAi control roots. In this analysis, no significant differences were found in inoculated PvNPC4-RNAi roots compared to inoculated RNAi control roots (10 dpi) (Fig 9A). Moreover, when comparing the abundance of the PvASL18a transcript in non-inoculated versus inoculated control roots, no significant difference was also observed. Likewise, PvASL18a transcript abundance did not change between non-inoculated and inoculated PvNPC4-RNAi roots (Fig 9A). On the other hand, PvASL18b transcript abundance did not change significantly in PvNPC4-RNAi inoculated roots compared to RNAi control roots (Fig 8B), as it did in non-inoculated roots (Fig 5B). Interestingly, PvASL18b transcript abundance in PvNPC4-RNAi inoculated roots significantly increased compared to PvNPC4-RNAi non-inoculated roots (Fig 9B).
Relative transcript abundance of PvASL18a (A) and PvASL18b (B). Ctrl-RNAi indicates transgenic roots carrying the control vector pTdT-SAC. PvNPC4-RNAi indicates transgenic roots carrying the PvNPC4 silencing construct (C1-RNAi and C8-RNAi lines). The lower and upper edges of the boxes delimit the first to third quartiles, respectively, the central horizontal line represents the median, and the whiskers indicate the maximum and minimum values in the data set. For statistical analysis, the Monte Carlo simulation test was used with 9999 samples without replacement (**** p ≤ 0.0001). Black dots in the box plots indicate individual samples from three biological replicates.
Discussion
The participation of non-specific phospholipases (NPCs) in plant development has not been thoroughly analyzed. Previous studies have mainly focused on Arabidopsis, where the role of NPC in root development was examined. Studies performed in other plant models as rice [19] and wheat (Triticum aestivum L,) [20] have limited their analysis to the expression of NPC during certain stages of development and abiotic stresses. Here, we showed that the common bean gene PvNPC4 plays a key role in root and nodule development.
PLC family in legumes
Our in silico analysis of the PLC family in common bean, L. japonicus, and barrelclover revealed that legume species generally possess larger numbers of PI-PLC members than NPC members, with the exception of L. japonicus, which has equal numbers in the two groups (S2 and S3 Table). This is in contrast to what has been reported in rice and wheat, two cereal species (Poaceae) that have more NPC members [19,20].
Protein sequence analysis revealed that the conserved domain characteristics of PI-PLCs and NPCs in plants [19,20] are preserved in common bean. In particular, all PvPI-PLCs in legumes retain the PLC_X, PLC_Y, and C2 domains. However, the EF-hand motif is not present in all proteins identified in this study, in agreement with the complement of PLCs in other species [19,20,56]. Importantly, the EF-hand motif was present in only four GmPI-PLCs, not in all GmPI-PLCs, as previously reported [22]. Regardless of gene structure, we observed nine exons in PvPI-PLC genes and three or four exons in PvNPC genes. This finding is in line with the number of exons found in many other species, including chickpea (Cicer arietinum L.) [56], soybean [22], rice [19], and wheat [20]. These findings suggest that the gene structure of both PLC subfamily genes has been conserved among divergent species.
The phylogenetic tree reconstructed here grouped PI-PLCs and NPCs into two separated clades, regardless of their originating species (Fig 1), consistent with other phylogenetic analyses [18,20,56]. The small phylogenetic distance observed between PvPLCs and GmPLCs (Fig 1) was also supported by a collinearity analysis (Fig 2). Notably, the phylogenetic distance between the PI-PLC clade and the NPC clade was greater than that between the NPC clade and the M. tuberculosis PLC used as an out-group. This observation is consistent with what was reported by Nakamura et al. (2005) who identified three conserved regions between in A. thaliana NPCs and a M. tuberculosis PLC.
Analysis of PvNPC4 function in root development
Analysis of NPC transcript abundance in Arabidopsis and rice has revealed that they vary by plant tissue and developmental stage [19,24]. We observed similar patterns for PvNPC4, whose transcript abundance fluctuated in roots between developmental stages (S9 Fig) and tissues, being the highest transcript abundance in roots (S10A Fig). This finding suggests a role of PvNPC4 in nodule development. Indeed, knockdown of PvNPC4 by RNAi shortened primary roots and resulted in fewer primary and lateral roots (Fig 4). These findings are consistent with the reduction in primary root length observed in two NPC knock-outs of npc3 and npc4 from A. thaliana [24].
A reduction of root length was also observed in NPC2 knock-down lines in A. thaliana in an npc6 knock-out background [25]. However, some of the NPC2 knock-down lines developed an increase in lateral root density, which contrasts with the reduction in lateral roots in PvNPC4-RNAi plants. Interestingly, exogenous supplementation of phosphocholine, a product of phosphatidylcholine hydrolysis by NPCs, restores normal root length [25], suggesting that phosphocholine may act as a signal molecule that regulates root development.
On the other hand, our results indicate that the abundance of transcripts of genes involved in auxin signaling was reduced by PvNPC4 silencing. Particularly, it was observed that the abundance of PvASL18b transcripts was reduced in PvNPC4 roots (Fig 5A). This gene encodes a transcriptional factor, LATERALORGAN BOUNDARIES-DOMAIN 16 (LBD16)/ASYMMETRIC LEAVES2-LIKE 18 (ASL18), that is activated by auxin in the founder cells of lateral roots [3]. In M. truncatula, lbd mutants showed a reduction in lateral root development of approximately 50%; in contrast, overexpression of an LBD16 gene induced the initiation of ectopic root primordia [55]. In addition, in L. japonicus, asl18a knock-out developed lower lateral roots density than wild-type plants [57]. Together, these findings suggest that the reduced root development in PvNPC4-RNAi composite plants are associated with a regulatory role of PvASL18b in auxin signaling.
The involvement of PvNPC4 in root development in an auxin-dependent manner is also supported by the reduced transcript abundance of the gene encoding the auxin transporter, PvPin1b, in PvNPC4-RNAi roots (Fig 5B). Phospholipases A2 are also involved in auxin transport by targeting the PIN protein to the plasma membrane. For instance, knockdown of PLA2 was shown to interfere with trafficking of the PIN protein to the plasma membrane in A. thaliana roots. Interestingly, the hydrolytic product of PLA2, lysophosphatidylethanolamine, restored PIN localization to the plasma membrane in a pla2α mutant [58]. These results evidence that lipid metabolism is crucial for the accurate auxin-dependent regulatory mechanism. Our findings suggest that PvNPC4 is a key regulator of root development by regulating auxin signaling.
Analysis of PvNPC4 function in nodule development
Legumes have developed different mechanisms to strictly control rhizobial symbiosis, with differential gene expression being essential [59–61]. In the present work, PvNPC4 transcript abundance showed a biphasic pattern being high at early stages of rhizobial symbiosis (24–72 hpi) and low at 14 dpi in inoculated roots and nodules compared to control (Fig 3). This pattern of PvNPC4 transcript abundance suggests its involvement in rhizobial infection and a putative negative effect on the later mature stages of symbiosis. The decline in PvNPC4 transcript abundance in inoculated roots and nodules at 14 dpi may reflect a mechanism to prevent phospholipid degradation once nodules have developed. It must be considered that NPCs have non-specific hydrolytic activity towards phospholipids [17]; therefore, their functions may be necessary in the early stages of symbiosis for lipid-mediated signaling that allows infection by rhizobia. At the later and mature stages, when nodules have formed, lipid turnover may be less active; consequently, the hydrolytic activity of PLCs may be more dispensable.
We found a significant decrease in the number of ITs in PvNPC4-RNAi roots, which was accompanied by fewer nodules (Fig 6). Previously, it was reported that PvRbohA silencing reduced PvEnod40 transcript abundance and ITs number in common bean [7]. Interesting, our results showed a reduced abundance of PvEnod40 transcripts in PvNPC4-RNAi inoculated roots (Fig 7B). Reduced ITs in common bean was earlier associated with PvEnod40 transcript abundance [60]; therefore, the drop in PvEnod40 transcript abundance in PvNPC4-RNAi roots may be involved in ITs development.
To further investigate a functional relationship between PvNPC4 and PvRbohA, PvRbohA transcript abundance was quantified in R. tropici-inoculated and non-inoculated PvNPC4-RNAi roots. The slight increase in PvRbohA transcript abundance due to PvNPC4 silencing, regardless of R. tropici inoculation (Fig 7C), suggests a role for PvNPC4 in regulating PvRbohA transcription. In this scenario it would be interesting to explore PvRbohA transcript abundance at other stages of the symbiosis in a PvNPC4 background. RBOHs are enzymes that produce superoxide anions, a reactive oxygen species (ROS) [62]; in particular, in common bean, ROS signaling has been shown to respond to rhizobial inoculation and NF perception [63,64]. Furthermore, the PvRbohA and PvRbohB genes in common bean play key roles during rhizobial symbiosis, as we have described previously [6,7].
ROS homeostasis is very sensitive to rhizobia inoculation and NF perception [63,64] and depending on its levels, can vary according to the stage of development of the symbiosis. Consequently, we suggest that the slight increase in PvRbohA transcript abundance observed in PvNPC4-RNAi roots may affect proper rhizobial infection. Intriguingly the transcript abundance of PvNIN, a master regulator of nodule organogenesis [2] that is also involved in IT formation [65], was not significantly affected by PvNPC4 silencing at 10 dpi (Fig 7A). However, considering the crucial role of NIN in regulating rhizobial symbiosis, its involvement in earlier stages of symbiosis is not discarded. Unfortunately, due to the marked reduction of root development, we had difficulty collecting root tissue at earlier stages.
In the case of the role of Enod40 in nodule organogenesis, this has been largely documented; for instance, it has been reported to regulate cortical cell division in soybean [66] and the number of nodules in peanut (Arachis hypogea L.) [67]. Therefore, the reduction in nodule number could be related to the decrease in PvEnod40 transcript abundance, in fact, PvEnod40 transcripts were undetectable in some samples. Considering the increase and decrease of PvNPC4 transcription in the early and late stages of the symbiosis, respectively, we asked whether the protein product of this gene is involved in the AON pathway. To explore this, transcript abundance of AON-related genes, such as RIC and TML, was assessed in inoculated and non-inoculate transgenic roots at 10 dpi. Our qPCR analysis shows that PvNPC silencing enhanced transcript abundance of PvRIC1 (Fig 8A). This gene has been shown to be transcriptionally activated by inoculation of R. phaseoli into common white bean [10], another variety of P. vulgaris, indicating an important role of the RIC1 protein in the AON mechanism in P. vulgaris.
RIC genes encode CLE peptides in response to rhizobia, initiating the AON. These peptides are transported through the xylem to the shoot where they are recognized by leucine-rich repeats receptor-like kinases (LRR-RLKs) that inhibit the production of miR2111. [54]. In M. truncatula miR2111 acts as a shoot-to-root signaling that negatively regulates nodule TML transcript abundance in roots, thereby negatively regulating nodule development [68]. In this scenario, the induction of PvRIC1 in inoculated PvNPC4-RNAi roots is consistent with the reduction of nodule number caused by PvNPC4 silencing (Fig 6B). Our results, together with the reported findings on the AON mechanism, suggest that PvNPC4 acts as a positive regulator of nodulation upstream RIC transcription.
The low transcript abundance of PvRIC2 in inoculated transgenic roots suggests that this gene is not induced immediately after R. tropici inoculation (10 dpi). However, it is intriguing that PvRIC2 transcript abundance increased in non-inoculated PvNPC4-RNAi roots and decreased in those inoculated with this difference being significant (Fig 8B). These observations point out that the PvNPC4 protein regulates PvRIC2 but, probably, at 10 dpi, PvRIC2 was not required for AON signaling, as was PvRIC1. Therefore, PvRIC2 reduction could be a compensatory mechanism of PvRIC transcription for the precise signaling function of AON.
On the other hand, a reduction of PvTMLa transcript abundance was observed in non-inoculated PvNPC4-RNAi roots, suggesting a role for this gene in the AON mechanism in common bean. Interestingly, this effect of PvNPC4 on PvTMLa transcription was not observed in inoculated roots (Fig. 8C). Interestingly, R. tropici inoculation induced a slight increase in PvTMLa transcript abundance in PvNPC4-RNAi and RNAi control inoculated roots compared to their non-inoculated counterparts, indicating the involvement of PvTMLa in the AON mechanism in common bean. However, the role of PvTMLa in the common bean-R. tropici symbiosis appears not to be regulated by PvNPC4 at 10 dpi. The common bean genome encodes five TML genes, where PvTMLa has the highest transcript abundance in inoculated roots and in nodules at 5 and 15 dpi (S13 Fig). This prompted us to explore PvTMLa transcription in inoculated and non-inoculated transgenic roots. The participation of the rest of the PvTML genes in the regulation of this symbiosis by PvNPC4 is of interest for future research. To our knowledge, this is the first exploration of the function of TML genes in common bean.
The LOB16/ASL18 genes have been shown to be involved in both root and nodule development [52,55]. Our results show that PvNPC4 silencing did not affect PvASL18a transcript abundance regardless of R. tropici inoculation (Fig 9A), suggesting a non-functional relationship of PvNPC4 and PvASL18a in root and nodule development. As observed in Fig 9B, PvASL18b seems to be involved in the role of PvNPC4 in root development, but not in nodule development.
Conclusions and perspectives
In the present study, we provide evidence that downregulation of PvNPC4 transcript abundance negatively affected root development in this legume species. Interestingly, this effect appears to be related to auxin transport, as the transcript abundance of genes involved in auxin signaling decreased significantly or slightly in PvNPC4-RNAi roots, depending on the gene analyzed. More detailed studies using auxin markers to elucidate dynamic changes in auxin transport and synthesis would be valuable for a better understanding of PvNPC4-mediated regulation of auxin-dependent root development. Further studies may be needed to examine the potential seedling lethality of PvNPC4 knockouts, as well as functional redundancy among PvNPC members.
The establishment of rhizobial symbiosis involves several biological processes, such as membrane turnover, lipid-mediated signaling and metabolism, and autoregulation of nodulation (AON). NPCs are enzymes closely involved in membrane remodeling and lipid metabolism due to their hydrolytic activity towards phospholipids. Furthermore, lipid signaling is a key signaling mechanism for the regulation of various functions in plants. We showed that downregulation of PvNPC4 decreased rhizobial infection in common bean hairy roots with a subsequent drop in nodule number. Importantly, our results show evidence for the involvement of PvNPC4 in the AON. To the best of our knowledge, this is the first report on the involvement of a PLC in rhizobial symbiosis.
In summary, we showed here that silencing PvNPC4 results in shorter and fewer roots, as well as fewer nodules. It is important to emphasize that a conserved regulatory pathway shared between lateral roots and nodule development has been suggested and discussed previously [3,57]. Therefore, we hypothesize that PvNPC4 might be involved in this shared pathway involving auxin signaling, as shown in the model illustrated in Fig 10.
(A) PvNPC is activated, in an unknown manner, in response to NFs secreted by rhizobia (green lines). The hydrolytic activity of PvNPC4 on phosphatidylcholine releases phosphocholine, which is suggested to act as a secondary messenger to induce the transcription of symbiosis-related genes that regulates rhizobial infection and nodule development. At the same time, transcription of auxin response genes is activated, which also regulates rhizobial symbiosis, depending on the concentration and location of auxin. When NPC4 expression is altered by RNAi interference (PvNPC4-RNAi in this work, red lines), or by another stimulus, the NPC4 protein level decreases; consequently, the concentration of phosphocholine decreases. Therefore, the positive role of NPC4 in regulating nodule development fails or is attenuated. Our results showed that under these conditions, the transcript abundance of PvRIC1 and PvEnod40 is modulated, which may regulate nodule development. (B) Under normal conditions, NPC plays a positive regulatory role in root development (green lines), probably mediated by phosphocholine. If NPC expression is decreased, as explained above (red lines), transcription of genes involved in auxin signaling, a master regulator of root development is inhibited. This would provoke a decrease in root development. Our results indicated that PvASL18b is involved in this mechanism. In summary, our results suggest that PvNPC4 modulates both root and nodule development by regulating auxin-mediated signaling (black dashed arrows).
Acknowledgments
We thank Ph. D. Citlali Fonseca-García for critical reading of the manuscript. We also thank B. Sc Mariel Escobar, B. Sc. Marlén Delgado, and B. Sc. Mary Jose Enriquez for their support in the experiments carried out in the greenhouse and Ph. D. Javier Montalvo-Arredondo for the support in image preparation. In addition, we thank Unidad de Síntesis y Secuenciación at Instituto de Biotecnología, UNAM for technical support regarding oligonucleotide synthesis and DNA sequencing.
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