Genome-wide identification and comparative analysis of the Amino Acid Transporter (AAT) gene family and their roles during Phaseolus vulgaris symbioses.

Amino acid transporters (AATs) are essential integral membrane proteins that serve multiple roles, such as facilitating the transport of amino acids across cell membranes. They play a crucial role in the growth and development of plants. Phaseolus vulgaris, a significant legume crop, serves as a valuable model for studying root symbiosis. In this study, we have conducted an exploration of the AAT gene family in P. vulgaris. In this research, we identified 84 AAT genes within the P. vulgaris genome sequence and categorized them into 12 subfamilies based on their similarity and phylogenetic relationships with AATs found in Arabidopsis and rice. Interestingly, these AAT genes were not evenly distributed across the chromosomes of P. vulgaris . Instead, there was an unusual concentration of these genes located toward the outer edges of chromosomal arms. Upon conducting motif analysis and gene structural analysis, we observed a consistent presence of similar motifs and an intron-exon distribution pattern among the subfamilies. When we analyzed the expression profiles of PvAAT genes, we noted tissue-specific expression patterns. Furthermore, our investigation into AAT gene expression under rhizobial and mycorrhizal symbiotic conditions revealed that certain genes exhibited high levels of expression. Specifically, ATLa5 and LHT2 was notably upregulated under both symbiotic conditions. These findings point towards a potential role of AATs in the context of rhizobial and mycorrhizal symbiosis in P. vulgaris, in addition to their well-established regulatory functions.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

Comprehensive Analysis of Phaseolus vulgaris SnRK Gene Family and Their Expression during Rhizobial and Mycorrhizal Symbiosis.

Sucrose non-fermentation-related protein kinase 1 (SnRK1) a Ser/Thr protein kinase, is known to play a crucial role in plants during biotic and abiotic stress responses by activating protein phosphorylation pathways. SnRK1 and some members of the plant-specific SnRK2 and SnRK3 sub-families have been studied in different plant species. However, a comprehensive study of the SnRK gene family in Phaseolus vulgaris is not available. Symbiotic associations of P. vulgaris with Rhizobium and/or mycorrhizae are crucial for the growth and productivity of the crop. In the present study, we identified PvSnRK genes and analysed their expression in response to the presence of the symbiont. A total of 42 PvSnRK genes were identified in P. vulgaris and annotated by comparing their sequence homology to Arabidopsis SnRK genes. Phylogenetic analysis classified the three sub-families into individual clades, and PvSnRK3 was subdivided into two groups. Chromosome localization analysis showed an uneven distribution of PvSnRK genes on 10 of the 11 chromosomes. Gene structural analysis revealed great variation in intron number in the PvSnRK3 sub-family, and motif composition is specific and highly conserved in each sub-family of PvSnRKs. Analysis of cis-acting elements suggested that PvSnRK genes respond to hormones, symbiosis and other abiotic stresses. Furthermore, expression data from databases and transcriptomic analyses revealed differential expression patterns for PvSnRK genes under symbiotic conditions. Finally, an in situ gene interaction network of the PvSnRK gene family with symbiosis-related genes showed direct and indirect interactions. Taken together, the present study contributes fundamental information for a better understanding of the role of the PvSnRK gene family not only in symbiosis but also in other biotic and abiotic interactions in P. vulgaris.

A protocol for the generation of Arachis hypogaea composite plants: A valuable tool for the functional study of mycorrhizal symbiosis.

PREMISE: Agrobacterium rhizogenes-induced hairy root systems are one of the most preferred and versatile systems for the functional characterization of genes. The use of hairy root systems is a rapid and convenient alternative for studying root biology, biotic and abiotic stresses, and root symbiosis in in vitro recalcitrant legume species such as Arachis hypogaea. METHODS AND RESULTS: We present a rapid, simplified method for the generation of composite A. hypogaea plants with transgenic hairy roots. We demonstrate a technique of hairy root induction mediated by A. rhizogenes from young A. hypogaea shoots. The efficacy of the system for producing transgenic roots is demonstrated using an enhanced green fluorescent protein (eGFP) expression vector. Furthermore, the application of the system for studying root branching is shown using the auxin-responsive marker DR5 promoter fused to ß-glucuronidase (GUS). Finally, the success of the hairy root system for root symbiotic studies is illustrated by inoculating hairy roots with arbuscular mycorrhizal fungi. CONCLUSIONS: In this study, we have developed a rapid, efficient, and cost-effective composite plant protocol for A. hypogaea that is particularly effective for root-related studies and for the validation of candidate genes in A. hypogaea during mycorrhizal symbiosis.

Exploration of Autophagy Families in Legumes and Dissection of the ATG18 Family with a Special Focus on Phaseolus vulgaris.

Macroautophagy/autophagy is a fundamental catabolic pathway that maintains cellular homeostasis in eukaryotic cells by forming double-membrane-bound vesicles named autophagosomes. The autophagy family genes remain largely unexplored except in some model organisms. Legumes are a large family of economically important crops, and knowledge of their important cellular processes is essential. Here, to first address the knowledge gaps, we identified 17 ATG families in Phaseolus vulgaris, Medicago truncatula and Glycine max based on Arabidopsis sequences and elucidated their phylogenetic relationships. Second, we dissected ATG18 in subfamilies from early plant lineages, chlorophytes to higher plants, legumes, which included a total of 27 photosynthetic organisms. Third, we focused on the ATG18 family in P. vulgaris to understand the protein structure and developed a 3D model for PvATG18b. Our results identified ATG homologs in the chosen legumes and differential expression data revealed the nitrate-responsive nature of ATG genes. A multidimensional scaling analysis of 280 protein sequences from 27 photosynthetic organisms classified ATG18 homologs into three subfamilies that were not based on the BCAS3 domain alone. The domain structure, protein motifs (FRRG) and the stable folding conformation structure of PvATG18b revealing the possible lipid-binding sites and transmembrane helices led us to propose PvATG18b as the functional homolog of AtATG18b. The findings of this study contribute to an in-depth understanding of the autophagy process in legumes and improve our knowledge of ATG18 subfamilies.

Target of rapamycin, PvTOR, is a key regulator of arbuscule development during mycorrhizal symbiosis in Phaseolus.

Target of rapamycin (TOR) is a conserved central growth regulator in eukaryotes that has a key role in maintaining cellular nutrient and energy status. Arbuscular mycorrhizal (AM) fungi are mutualistic symbionts that assist the plant in increasing nutrient absorption from the rhizosphere. However, the role of legume TOR in AM fungal symbiosis development has not been investigated. In this study, we examined the function of legume TOR in the development and formation of AM fungal symbiosis. RNA-interference-mediated knockdown of TOR transcripts in common bean (Phaseolus vulgaris) hairy roots notably suppressed AM fungus-induced lateral root formation by altering the expression of root meristem regulatory genes, i.e., UPB1, RGFs, and sulfur assimilation and S-phase genes. Mycorrhized PvTOR-knockdown roots had significantly more extraradical hyphae and hyphopodia than the control (empty vector) roots. Strong promoter activity of PvTOR was observed at the site of hyphal penetration and colonization. Colonization along the root length was affected in mycorrhized PvTOR-knockdown roots and the arbuscules were stunted. Furthermore, the expression of genes induced by AM symbiosis such as SWEET1, VPY, VAMP713, and STR was repressed under mycorrhized conditions in PvTOR-knockdown roots. Based on these observations, we conclude that PvTOR is a key player in regulating arbuscule development during AM symbiosis in P. vulgaris. These results provide insight into legume TOR as a potential regulatory factor influencing the symbiotic associations of P. vulgaris and other legumes.

Differential tetraspanin genes expression and subcellular localization during mutualistic interactions in Phaseolus vulgaris.

Arbuscular mycorrhizal fungi and rhizobia association with plants are two of the most successful plant-microbe associations that allow the assimilation of P and N by plants, respectively. These mutualistic interactions require a molecular dialogue, i.e., legume roots exude flavonoids or strigolactones which induce the Nod factors or Myc factors synthesis and secretion from the rhizobia or fungi, respectively. These Nod or Myc factors trigger several responses in the plant root, including calcium oscillations, and reactive oxygen species (ROS). Furthermore, superoxide and H2O2 have emerged as key components that regulate the transitions from proliferation to differentiation in the plant meristems. Similar to the root meristem, the nodule meristem accumulates superoxide and H2O2. Tetraspanins are transmembrane proteins that organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking and ROS generation. Plant tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, and hormonal regulation and recently have been reported as a specific marker of exosomes in animal and plant cells and key players at the site of plant fungal infection. In this study, we conducted transcriptional profiling of the tetraspanin family in common bean (Phaseolus vulgaris L. var. Negro Jamapa) to determine the specific expression patterns and subcellular localization of tetraspanins during nodulation or under mycorrhizal association. Our results demonstrate that the tetraspanins are transcriptionally modulated during the mycorrhizal association, but are also expressed in the infection thread and nodule meristem development. Subcellular localization indicates that tetraspanins have a key role in vesicular trafficking, cell division, and root hair polar growth.

Cysteine-Rich Receptor-Like Kinase Gene Family Identification in the Phaseolus Genome and Comparative Analysis of Their Expression Profiles Specific to Mycorrhizal and Rhizobial Symbiosis.

Receptor-like kinases (RLKs) are conserved upstream signaling molecules that regulate several biological processes, including plant development and stress adaptation. Cysteine (C)-rich receptor-like kinases (CRKs) are an important class of RLK that play vital roles in disease resistance and cell death in plants. Genome-wide analyses of CRK genes have been carried out in Arabidopsis and rice, while functional characterization of some CRKs has been carried out in wheat and tomato in addition to Arabidopsis. A comprehensive analysis of the CRK gene family in leguminous crops has not yet been conducted, and our understanding of their roles in symbiosis is rather limited. Here, we report the comprehensive analysis of the PhaseolusCRK gene family, including identification, sequence similarity, phylogeny, chromosomal localization, gene structures, transcript expression profiles, and in silico promoter analysis. Forty-six CRK homologs were identified and phylogenetically clustered into five groups. Expression analysis suggests that PvCRK genes are differentially expressed in both vegetative and reproductive tissues. Further, transcriptomic analysis revealed that shared and unique CRK genes were upregulated during arbuscular mycorrhizal and rhizobial symbiosis. Overall, the systematic analysis of the PvCRK gene family provides valuable information for further studies on the biological roles of CRKs in various Phaseolus tissues during diverse biological processes, including Phaseolus-mycorrhiza/rhizobia symbiosis.

RbohA coordinates lateral root emergence in common bean.

Respiratory burst oxidase homologs (RBOHs) constitute a multigene family in plants. These reactive oxygen species (ROS)-generating enzymes participate in diverse biological processes. We previously demonstrated that PvRbohB plays an important role in lateral root (LR) development in Phaseolus vulgaris. However, little is known about the roles of other Rboh members in root development. Here, we report that PvRbohA is involved in LR emergence in P. vulgaris. PvRbohA was expressed in wild-type P. vulgaris root tissues, e.g., the radicle, inter LR zone, and LR zone, and its expression increased during LR formation. Analysis of the spatio-temporal expression patterns of a reporter construct under the control of the PvRbohA promoter (PvRbohA::GUS-GFP) in transgenic roots showed that PvRbohA was active at all three stages of LR development, but its spatial expression pattern varied at each stage. The relative expression levels of PvRbohA during LR formation correlated with the activity of PvRbohA promoter. Furthermore, upon PvRbohA transcript silencing, LR growth was significantly altered in transgenic hairy roots. These findings suggest that RbohA participates in LR initiation, emergence, and development in the legume P. vulgaris by delimiting the region for LR emergence.

In BPS1 Downregulated Roots, the BYPASS1 Signal Disrupts the Induction of Cortical Cell Divisions in Bean-Rhizobium Symbiosis.

BYPASS1 (BPS1), which is a well-conserved gene in plants, is required for normal root and shoot development. In the absence of BPS1 gene function, Arabidopsis overproduces a mobile signalling compound (the BPS1 signal) in roots, and this transmissible signal arrests shoot growth and causes abnormal root development. In addition to the shoot and root meristem activities, the legumes also possess transient meristematic activity in root cortical cells during Rhizobium symbiosis. We explored the role of Phaseolus vulgaris BPS1 during nodule primordium development using an RNA-interference (RNAi) silencing approach. Our results show that upon Rhizobium infection, the PvBPS1-RNAi transgenic roots failed to induce cortical cell divisions without affecting the rhizobia-induced root hair curling and infection thread formation. The transcript accumulation of early nodulin genes, cell cyclins, and cyclin-dependent kinase genes was affected in RNAi lines. Interestingly, the PvBPS1-RNAi root nodule phenotype was partially rescued by exogenous application of fluridone, a carotenoid biosynthesis inhibitor, which was used because the carotenoids are precursors of BPS1 signalling molecules. Furthermore, we show that the PvBPS1 promoter was active in the nodule primordia. Together, our data show that PvBPS1 plays a vital role in the induction of meristematic activity in root cortical cells and in the establishment of nodule primordia during Phaseolus-Rhizobium symbiosis.

Respiratory Burst Oxidase Homolog Gene A Is Crucial for Rhizobium Infection and Nodule Maturation and Function in Common Bean.

Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA-I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris-Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

Differentially expressed genes in mycorrhized and nodulated roots of common bean are associated with defense, cell wall architecture, N metabolism, and P metabolism.

Legumes participate in two important endosymbiotic associations, with phosphorus-acquiring arbuscular mycorrhiza (AM, soil fungi) and with nitrogen-fixing bacterial rhizobia. These divergent symbionts share a common symbiotic signal transduction pathway that facilitates the establishment of mycorrhization and nodulation in legumes. However, the unique and shared downstream genes essential for AM and nodule development have not been identified in crop legumes. Here, we used ion torrent next-generation sequencing to perform comparative transcriptomics of common bean (Phaseolus vulgaris) roots colonized by AM or rhizobia. We analyzed global gene expression profiles to identify unique and shared differentially expressed genes (DEGs) that regulate these two symbiotic interactions, and quantitatively compared DEG profiles. We identified 3,219 (1,959 upregulated and 1,260 downregulated) and 2,645 (1,247 upregulated and 1,398 downregulated) unigenes that were differentially expressed in response to mycorrhizal or rhizobial colonization, respectively, compared with uninoculated roots. We obtained quantitative expression profiles of unique and shared genes involved in processes related to defense, cell wall structure, N metabolism, and P metabolism in mycorrhized and nodulated roots. KEGG pathway analysis indicated that most genes involved in jasmonic acid and salicylic acid signaling, N metabolism, and inositol phosphate metabolism are variably expressed during symbiotic interactions. These combined data provide valuable information on symbiotic gene signaling networks that respond to mycorrhizal and rhizobial colonization, and serve as a guide for future genetic strategies to enhance P uptake and N-fixing capacity to increase the net yield of this valuable grain legume.

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter ß-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates ≥2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.

The target of rapamycin (TOR) protein kinase regulates metabolism, growth, and life span in yeast, animals, and plants in coordination with nutrient status and environmental conditions. The nutrient-dependent nature of TOR functionality makes this kinase a putative regulator of symbiotic associations involving nutrient acquisition. However, TOR's role in these processes remains to be understood. Here, we uncovered the role of TOR during the bean (Phaseolus vulgaris)-Rhizobium tropici (Rhizobium) symbiotic interaction. TOR was expressed in all tested bean tissues, with higher transcript levels in the root meristems and senesced nodules. We showed TOR promoter expression along the progressing infection thread and in the infected cells of mature nodules. Posttranscriptional gene silencing of TOR using RNA interference (RNAi) showed that this gene is involved in lateral root elongation and root cell organization and also alters the density, size, and number of root hairs. The suppression of TOR transcripts also affected infection thread progression and associated cortical cell divisions, resulting in a drastic reduction of nodule numbers. TOR-RNAi resulted in reduced reactive oxygen species accumulation and altered CyclinD1 and CyclinD3 expression, which are crucial factors for infection thread progression and nodule organogenesis. Enhanced expression of TOR-regulated ATG genes in TOR-RNAi roots suggested that TOR plays a role in the recognition of Rhizobium as a symbiont. Together, these data suggest that TOR plays a vital role in the establishment of root nodule symbiosis in the common bean.

An Autophagy-Related Kinase Is Essential for the Symbiotic Relationship between Phaseolus vulgaris and Both Rhizobia and Arbuscular Mycorrhizal Fungi.

Eukaryotes contain three types of lipid kinases that belong to the phosphatidylinositol 3-kinase (PI3K) family. In plants and Saccharomyces cerevisiae, only PI3K class III family members have been identified. These enzymes regulate the innate immune response, intracellular trafficking, autophagy, and senescence. Here, we report that RNAi-mediated downregulation of common bean (Phaseolus vulgaris) PI3K severely impaired symbiosis in composite P. vulgaris plants with endosymbionts such as Rhizobium tropici and Rhizophagus irregularis Downregulation of Pv-PI3K was associated with a marked decrease in root hair growth and curling. Additionally, infection thread growth, root-nodule number, and symbiosome formation in root nodule cells were severely affected. Interestingly, root colonization by AM fungi and the formation of arbuscules were also abolished in PI3K loss-of-function plants. Furthermore, the transcript accumulation of genes encoding proteins known to interact with PI3K to form protein complexes involved in autophagy was drastically reduced in these transgenic roots. RNAi-mediated downregulation of one of these genes, Beclin1/Atg6, resulted in a similar phenotype as observed for transgenic roots in which Pv-PI3K had been downregulated. Our findings show that an autophagy-related process is crucial for the mutualistic interactions of P. vulgaris with beneficial microorganisms.

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

BACKGROUND: Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. RESULTS: Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. CONCLUSIONS: We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.

Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation.

Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant's response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis.

Co-overexpression of Brassica juncea NPR1 (BjNPR1) and Trigonella foenum-graecum defensin (Tfgd) in transgenic peanut provides comprehensive but varied protection against Aspergillus flavus and Cercospora arachidicola.

KEY MESSAGE: Coexpression of two antifungal genes ( NPR1 and defensin ) in transgenic peanut results in the development of resistance to two major fungal pathogens, Aspergillus flavus and Cercospora arachidicola. Fungal diseases have been one of the principal causes of crop losses with no exception to peanut (Arachis hypogeae L.), a major oilseed crop in Asia and Africa. To address this problem, breeding for fungal disease resistance has been successful to some extent against specific pathogens. However, combating more than one fungal pathogen via breeding is a major limitation in peanut. In the present study, we demonstrated the potential use of co-overexpression of two genes, NPR1 and defensin isolated from Brassica juncea and Trigonella foenum-graecum respectively; that offered resistance towards Aspergillus flavus in peanut. The transgenic plants not only resisted the mycelial growth but also did not accumulate aflatoxin in the seeds. Resistance was also demonstrated against another pathogen, Cercospora arachidicola at varied levels; the transgenic plants showed both reduction in the number of spots and delay in the onset of disease. PCR, Southern and Western blot analysis confirmed stable integration and expression of the transgenes in the transgenic plants. The combinatorial use of the two pathogen resistance genes presents a novel approach to mitigate two important fungal pathogens of peanut.

Effect of Rhizobium and arbuscular mycorrhizal fungi inoculation on electrolyte leakage in Phaseolus vulgaris roots overexpressing RbohB.

Respiratory oxidative burst homolog (RBOH)-mediated reactive oxygen species (ROS) regulate a wide range of biological functions in plants. They play a critical role in the symbiosis between legumes and nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi. For instance, overexpression of PvRbohB enhances nodule numbers, but reduces mycorrhizal colonization in Phaseolus vulgaris hairy roots and downregulation has the opposite effect. In the present study, we assessed the effect of both rhizobia and AM fungi on electrolyte leakage in transgenic P. vulgaris roots overexpressing (OE) PvRbohB. We demonstrate that elevated levels of electrolyte leakage in uninoculated PvRbohB-OE transgenic roots were alleviated by either Rhizobium or AM fungi symbiosis, with the latter interaction having the greater effect. These results suggest that symbiont colonization reduces ROS elevated electrolyte leakage in P. vulgaris root cells.

RbohB, a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization.

The reactive oxygen species (ROS) generated by respiratory burst oxidative homologs (Rbohs) are involved in numerous plant cell signaling processes, and have critical roles in the symbiosis between legumes and nitrogen-fixing bacteria. Previously, down-regulation of RbohB in Phaseolus vulgaris was shown to suppress ROS production and abolish Rhizobium infection thread (IT) progression, but also to enhance arbuscular mycorrhizal fungal (AMF) colonization. Thus, Rbohs function both as positive and negative regulators. Here, we assessed the effect of enhancing ROS concentrations, by overexpressing PvRbohB, on the P. vulgaris--rhizobia and P. vulgaris--AMF symbioses. We estimated superoxide concentrations in hairy roots overexpressing PvRbohB, determined the status of early and late events of both Rhizobium and AMF interactions in symbiont-inoculated roots, and analyzed the nodule ultrastructure of transgenic plants overexpressing PvRbohB. Overexpression of PvRbohB significantly enhanced ROS production, the formation of ITs, nodule biomass, and nitrogen-fixing activity, and increased the density of symbiosomes in nodules, and the density and size of bacteroides in symbiosomes. Furthermore, PvCAT, early nodulin, PvSS1, and PvGOGAT transcript abundances were elevated in these nodules. By contrast, mycorrhizal colonization was reduced in roots that overexpressed RbohB. Overexpression of PvRbohB augmented nodule efficiency by enhancing nitrogen fixation and delaying nodule senescence, but impaired AMF colonization.