Plants, just like any other living organism, naturally get attacked by various pathogenic microorganisms such as bacteria, fungi and viruses. However, unlike animals that utilize their specialized circulatory macrophage system to protect themselves, plants instead use a multi-layered complex system termed the plant innate immunity, which recognizes pathogens and transducing downstream defense responses. They have developed a unique type of transmembrane receptors or R proteins, which extracellularly, are capable of recognizing pathogen-associated molecular patterns (PAMP) such as flagellin and chitin, while intracellularly, they activate their harbored nucleotide cyclases (NCs) such as adenylyl cyclases (ACs), to generate second messenger molecules such as 3',5'-cyclic adenosine monophosphate (cAMP), which then propagates and magnifies the defense response. To date, only a single R protein from Arabidopsis thaliana (AtLRR) has been shown to possess AC activity as well as having the ability to defend plants against infection by biotrophic and hemi-biotrophic pathogens. Therefore, in order to further broaden information around the functional roles of this protein (AtLRR), we explored it further, using an array of web-based tools or bioinformatics. These included structural analysis, anatomical expression analysis, developmental expression analysis, co-expression analysis, functional enrichment analysis, stimulusspecific expression analysis and promoter analysis. Findings from structural analysis showed that AtLRR is a multi-domain, trans-membrane molecule that is multi-functional, and thus consistent with all known R-proteins.
Downstream signalling involving adenylyl cyclases (ACs) and kinases is a key component of several processes in plants including cell division, growth, and response to stress. ACs are enzymes that generate the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP) while kinases are enzymes that catalyze the addition of a phosphate group to other molecules (trans-phosphorylation) or themselves (auto-phosphorylation). Apparently, while there has been an expanded record of various ACs and kinases identified in plants, no plant molecule to date has been shown to possess both the AC and kinase activities/functions and with such activities/functions having the characteristic of cross-talking interactions. Therefore, in an endeavor to find such a molecule, we searched the amino acid sequence of a known Arabidopsis AC, pentatricopeptide repeat (AtPPR) protein, and found a kinase-specific sequence signature (KSSS), which we speculated to be working in synergy with the AC center in this protein during downstream signalling. So, in order to test if this additional center is catalytically active and perhaps also having some cross-talking interactions with the AC center, we cloned, expressed, and affinity purified a truncated version of AtPPR, harboring both the AC and KSSS centers (AtPPR-AC/K). When tested in vitro, the recombinant AtPPR-AC/K showed a Mn2+-dependent AC activity that is positively enhanced by Ca2+ and HCO3− and a trans-/auto-phosphorylation kinase activity capable of utilizing both ATP and GTP as substrates and specific to the serine, threonine, and tyrosine amino acids as target residues. In addition, the kinase activity of AtPPR-AC/K was found to be reduced by cAMP while at the same time, it was totally shut down by Ca2+. This thus qualified both cAMP and Ca2+ as molecular switches or modulators, capable of regulating AtPPR functions through cross-talking interactions between the activities of its two domains. Our work, therefore, has essentially established AtPPR as the first member of a new class of moonlighting proteins with AC and kinase activities that have cross-talking interactions between themselves, conceivably presenting this protein as an ideal candidate for further explorations to improve plants, particularly agricultural crops.
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