In nature, plants are attacked by various pathogenic microorganisms that include bacteria, fungi and nematodes [1]. However, unlike animals that utilize their specialized circulatory macrophage system to protect themselves, plants instead use a multi-layered complex system termed plant innate immunity, that recognizes pathogens and transducing downstream defense responses [1]. Ideally, this plant innate immune system is divided into two; the first-line or basal immunity and the second-line immunity [1] [2].

First-line or basal immunity comes into action when pathogen/microbial-associated molecular patterns (PAMPs/MAMPs) such as the bacterial flagellin, fungal lipopolysaccharide or oomycetic cellulose binding elicitor proteins are recognized extracellularly by plant transmembrane receptors, termed pattern recognition receptors (PRRs) [2] [3]. Once triggered, the PRRs catalyze the production of cyclic nucleotide monophosphates (cNMP) i.e., 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guanosine monophosphate (cGMP) at their cytosolic end, which then facilitate entry of Ca²⁺ ions through the cyclic nucleotide gated ion channels (CNGCs) [4]. The increase in cytosolic Ca²⁺ ion concentration is an important primary event in pathogen signalling, that triggers downstream innate immune responses [5]. As the free cytosolic Ca²⁺ increases, the amount of Ca²⁺ bound to calmodulin (CaM) or calmodulin-like (CML) protein also increases, which then triggers the synthesis of downstream signalling components such as nitric oxide (NO) and hydrogen peroxide (H₂O₂), essential for the initiation and development of the hypersensitive response (HR) [5]. HR is essentially a collection of plant defense responses against pathogen infection that leads to rapid programmed cell death (PCD) of cells surrounding the pathogen-infected area, to prevent the spread of a disease [5]. The first-line or basal immunity is also known as the PAMPs-triggered immunity (PTI) [2] [3].

Second line immunity comes into action when the first-line immunity has been evaded, and it involves some highly specific cognate disease resistance (R) proteins that either directly or indirectly recognize pathogen effector proteins [6]. This type of immunity is also known as the effector-triggered immunity (ETI). Most R genes encode proteins that contain a nucleotide binding site (NBS) and leucine rich repeats (LRRs) or simply NBS-LRR proteins [2]. Pathogen effector molecules e.g., the AvrPto molecules of Pseudomonas syringae, typically alter the structure of NBS-LRR proteins through a direct binding or modification of other host plant proteins and allowing an exchange of ADP for ATP. The binding of ATP to the NBS domain then results in the activation of a signal transduction system through creation of binding sites for downstream signalling molecules and formation of central base binding (CBB) protein multimers. The dissociation of the pathogen effector proteins and modified effector targets from the NBS domain then results in the hydrolysis of ATP and a return of the NBS-LRR protein to its original inactive state [2] [7].

To this day, only a single NBS-LRR protein, AtLRR encoded by the At3g14460 gene in Arabidopsis thaliana, has been experimentally established to be an adenylyl cyclase (AC) [8] [9] with a role in defense response against the biotrophic fungus, Golovinomyces orontii and the hemi-biotrophic bacteria, Pseudomonas syringae [8]. With respect to AC activity, AtLRR was shown to display a multi-domain in vitro activity that is Mn²⁺-dependent and stimulated by Ca²⁺ while at the same time, the protein could rescue AC-deficiency in a mutant (cyaA) Escherichia coli or SP850 strain [8] [9]. With regard to disease resistance, knock-out mutants of AtLRR were found to have compromised immune responses to G. orontii and P. syringae but not against Botrytis cinerea, which is a necrotrophic fungus [8]. Therefore, in this reported study, we focused on using web-based tools or bioinformatics to elaborate further on the functional roles of this protein molecule both as an AC and NBS-LRR. Ideally, bioinformatics is simply defined as the science of data management systems in the genomics and proteomics of life forms, whereby biology, computer science and information technology merge into a single discipline.

Our work therefore, was motivated by the fact that since in nature proteins do work as a team or network to achieve common biological functions, as a result, bioinformatics can then be used as a tool with the power to both unravel and predict important information such as structure, solubility, interactions and functions of unknown and/or uncharacterized. In this study therefore, a combination of structural analysis, anatomical expression analysis, developmental expression analysis, co-expression analysis, functional enrichment analysis, stimulus-specific expression analysis and promoter analysis of the AtLRR protein was undertaken in selected mutant and wild type lines to circumscribe and further elaborate its function.

In plants, basal or first line immunity against pathogens provides a pre-infection resistance layer, that involves recognition of conserved structural components of pathogen such as flagellin or chitin, also referred to as pathogen-associated molecular patterns (PAMPs), ultimately leading to PAMP-triggered immunity (PTI) [42] [43]. A second layer of the plant defense system involves intracellular receptors that are products of the resistance (R) genes. These receptors recognize products of pathogen avirulence (Avr) genes, leading to rapid activation of defense responses such as the hypersensitive response (HR) at the infection sites. This layer of defense is often referred as effector-triggered immunity (ETI) [42] [43]. R genes encode proteins containing a nucleotide binding site (NBS) and leucine rich repeats (LRRs) or simply NBS-LRR proteins [2].

In Arabidopsis thaliana, there are approximately 150 NBS-LRR encoding genes, including At3g14460, that codes for an AtLRR protein [44]. This AtLRR protein was recently established to be an adenylyl cyclase (AC) [8] [9] with a role in resistance to infection by biotrophs and hemi-biotrophs [8] . ACs are enzymes capable of catalyzing the conversion of adenosine 5’-triphosphate (ATP) to the second messenger molecule, 3’,5’-cyclic adenosine monophosphate (cAMP) [45] [46] [47]. cAMP in turn, controls various downstream plant processes such as the cell cycle [48], growth of pollen tubes [29] [30] [31], and responses to biotic and abiotic stress [49] [50] [51]. On the other hand, biotrophs and hemi-biotrophs are obligate pathogens that establish a close and long-term nutritional relationship with their host cells and continuously absorb nutrients without causing damage to cells [42] [43]. These pathogens further trigger the onset of the salicylic acid (SA) dependent signalling system, which then stimulates and controls the establishment of systemic acquired resistance (SAR) in plants [52].

In this study, our analysis of the structural features of AtLRR, showed that the protein is a trans-membrane multi-domain molecule (Figure 1), capable of performing multiple functions e.g., AC activity [8] [9] and disease resistance [8]. These observed structural features of AtLRR, make it an ideal candidate for the PTI and ETI, whereby it effects the recognition of the pathogen effector molecules with its extracellular domain and then instigate downstream disease resistance processes via its intracellular domain [2].

From the anatomical and developmental expression profiles of AtLRR, it was noted that the protein was mostly expressed in pollen grains and flowers, senescing leaves as well as during the development of seeds, shoots, roots, seedlings, leaves, flowers, and siliques (Figure 2 and Figure 3). This was worth noting because it directly implicated AtLRR into plant reproduction, plant defense and plant development respectively—three key processes that are very important to plants. Apparently, the noted implication of AtLRR in plant reproduction, particularly as an AC, is literally not unusual because of three reasons. Firstly, cAMP has been shown to be directly involved in the growth and re-orientation of pollen tubes in Liliumlongiflorum [29], Agapanthus umbellatus [30], Zeamays [31] and A. thaliana [32]. Secondly, cAMP has been shown to regulate the development of male reproductive organs in Marchantia polymorpha [53]. Thirdly and lastly, cAMP has been shown to regulate flowering in Lemma gibba [54]. Once more, the noted implication of AtLRR in leaf senescence is also not unusual as it directly links AtLRR, both as an AC and disease resistance protein, to signal interactions that normally take place between pathogens and host plants during defense responses [55]. In this interaction, biotrophs often delay senescence to keep host cells alive, and resistance in this case is then achieved by senescence-like processes in the host while on the other hand, necrotrophs promote senescence in the host, and preventing early senescence would then be the resistance strategy for plants [55]. Hemi-biotrophs are involved in both patterns [55]. Lastly, the noted implication of AtLRR, again as an AC, in plant development is also not unusual because cAMP was previously shown to control the cell cycle in tobacco [48].

Further analysis of the expression profile of AtLRR showed that its gene (At3g14460) is mostly co-expressed and co-regulated with several other genes linked to disease resistance and cAMP signalling (Table 1). Of interest are At4g19520, At3g14470, At4g14610, At5g46470, At5g45000, At5g05190 and At1g72950, which all code for disease resistance proteins and At5g49680 that encodes a kinky pollen protein responsible for pollen tube growth (reproduction) and root hair cell tip growth (development). Once again, this is in tandem with the recently confirmed activities of AtLRR as an AC and disease resistance protein [8] [9].

When we extended our analysis to identify conditions that induce the expression of At3g14460 and its correlated genes (ECGG50) (Table 1), we noted very strong induction by the hemi-biotrophic pathogens, Pseudomonas syringae [33] and Phytophthora parasitica [34] [35], and their associated effector molecule, flagellin 22 [36] or its synthetic analogues, DFPM ([5-(3,4-dichlorophenyl)-2-furanyl]-1-piperidinyl-methanethione) and CPM (chlorphenamine) [37] (Figure 4). There was also strong induction in response to abscisic acid (ABA) (Figure 4)—a phytohormone that is chiefly involved in leaf senescence [55]. Induction by P. syringae and P. parasitica is consistent with the recently reported upregulation of the expression of AtLRR in response to the powdery mildew fungus Golovinomyces orontii, which is a biotroph and the Gram-negative bacterium P. syringae that is a hemi-biotroph [8]. In addition, ACs and cAMP have previously been directly implicated in disease resistance in various plants that include Nicotiana benthamiana [56], Hippeastrum hybridum [57], A. thaliana [50] and Brachypodium distachyon [58]. Thus, these findings are therefore, very consistent with the established role of AtLRR in plant cAMP-dependent signal transduction pathways against the biotrophic and hemi-biotrophic pathogens [52].

In addition, when we further subjected At3g14460 and its correlated genes (ECGG50) (Table 1) to promoter enrichment analysis, we found out that this gene together with its co-expressed partners, have a common transcription factor binding site (TFBS) in their promoters (Figure 5) that then allows them to be co-regulated and ultimately co-function [59]. The identified common TFBS is the W-box hexamer, TTGACY core element, known to bind WRKY transcription factors (TFs) [39] [40]. In Arabidopsis, the WRKY TF superfamily consists of 74 members [40] that are known to play significant roles in the transcriptional reprogramming associated with plant responses to pathogens and SA-signalling [40].

This study, therefore, has managed to explore around the functional roles of AtLRR both as an AC and disease resistance protein and elaborated such an exploration. The study has unequivocally managed to establish that, as an AC and disease resistance protein, AtLRR has key roles in plant disease resistance (against the biotrophic and hemi-biotrophic pathogens), plant reproduction (flowering, pollen tube growth and re-orientation, and development of male reproductive organs) and plant development (the cell cycle, and tissue and organ development). The protein effects its functions via a W-box or WRKY transcription factor, TTGACY, mediated by cAMP.

Considering the significance and importance of the processes in which AtLRR is involved, it is crucial that this novel protein candidate is studied further to understand its exact mechanisms of action and perhaps for the possible generation of some disease resistant crops/strong cultivars in the agricultural and horticultural sectors.

This work was funded by North-West University (NWU) and the National Research Foundation (NRF) of South Africa (Grant Numbers: CSUR78843 & CSUR93635).

OR and TN conceived and designed the study; AS-M did the experimental analyses; TBD, KSS, EDB and NMM facilitated the experimental analyses; MMT and DTK prepared the heat map. All authors contributed to the writing of the manuscript and approved the final version.

The authors declare no conflict of interest.

Sibanda-Makuvise, A., Dikobe, T.B., Sehlabane, K.S., Bobo, E.D., Mametja, N.M., Takundwa, M.M., Kawadza, D.T., Ncube, T. and Ruzvidzo, O. (2022) Elaborating the Functional Roles of a Leucine-Rich Repeat Protein from Arabidopsis thaliana. American Journal of Plant Sciences, 13, 1381-1401. https://doi.org/10.4236/ajps.2022.1311094