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. 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. 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.

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 Phaseolus CRK 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.

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.

Nitrogen is an essential macronutrient critical for plant growth and productivity. Plants have the capacity to uptake inorganic nitrate and ammonium, with nitrate playing a crucial role as a signaling molecule in various cellular processes. The availability of nitrate and the signaling pathways involved finely tune the processes of nitrate uptake and assimilation. NIN-like proteins (NLPs), a group of transcription factors belonging to the RWP-RK gene family, act as major nitrate sensors and are implicated in the primary nitrate response (PNR) within the nucleus of both non-leguminous and leguminous plants through their RWP-RK domains. In leguminous plants, NLPs are indispensable for the initiation and development of nitrogen-fixing nodules in symbiosis with rhizobia. Moreover, NLPs play pivotal roles in plant responses to abiotic stresses, including drought and cold. Recent studies have identified NLP homologs in oomycete pathogens, suggesting their potential involvement in pathogenesis and virulence. This review article delves into the conservation of RWP-RK genes, examining their significance and implications across different plant species. The focus lies on the role of NLPs as nitrate sensors, investigating their involvement in various processes, including rhizobial symbiosis in both leguminous and non-leguminous plants. Additionally, the multifaceted functions of NLPs in abiotic stress responses, developmental processes, and interactions with plant pathogens are explored. By comprehensively analyzing the role of NLPs in nitrate signaling and their broader implications for plant growth and development, this review sheds light on the intricate mechanisms underlying nitrogen sensing and signaling in various plant lineages.

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.

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 β‐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.