Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP261–379) can produce cyclic adenosine 3′,5′ monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization.

Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5'-triphosphate to cyclic 3', 5'--adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.

Background The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus (SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L. Methods Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 h post-transfection. Relative SACMV DNA accumulation was determined via qPCR using DpnI-digested total DNA, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2−ΔΔCTstatistical method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model. Results The differential expression of unedited and mutant MeE3L during SACMV infection of model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed that SACMV DNA accumulation in cassava protoplasts is genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGless MeE3Lwhich is silenced by SACMV-induced mutations. SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces. Conclusions This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in planta functional and interaction studies.

Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC 261-388 ) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule.From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC 261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC 261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.

Background Second messengers have a key role in linking environmental stimuli to cellular responses. One such messenger, 3′,5′-cyclic adenosine monophosphate (cAMP) generated by adenylyl cyclase (AC), has long been established as an essential signaling molecule in many physiological processes of higher plants, including growth, development and stress response. To date, very few ACs have been identified in plants, thus a need to search for more. Objective To test the probable AC activity of an Arabidopsis MEE (AtMEE) protein and infer its function bioinformatically. Methods A truncated version of the AtMEE protein (encoded by At2g34780 gene) harboring the annotated AC catalytic center (AtMEE-AC) was cloned and expressed in BL21 Star pLysS Escherichia coli cells followed by its purification using the nickel-nitriloacetic acid (Ni-NTA) affinity system. The purified protein was tested for its probable in vitro AC activity by enzyme immunoassay. The AtMEE-AC protein was also expressed in the SP850 mutant E. coli strain, followed by assessment (visually) of its ability to complement the AC-deficiency (cyaA mutation) in this mutant. Finally, the AtMEE protein was analyzed bioinformatically to infer its probable biological function(s). Results AtMEE is an AC molecule, whose in vitro activity is Mn2+-dependent and positively modulated by NaF. Moreover, AtMEE is capable of complementing the AC-deficiency (cyaA) mutation in the SP850 mutant strain. AtMEE is primarily involved in embryo development and also specifically expressed in response to abiotic stress via the MYB expression core motif signaled by cAMP. Conclusion 
AtMEE is an AC protein, whose functions are associated with embryo development and response to abiotic stress.

Adenylyl cyclase (AC) is an enzyme that catalyses the formation of the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP). cAMP, in turn, regulates key physiological processes such as cell division, growth, reproduction, development and response to stress. However, while cAMP is increasingly becoming an important signalling molecule in higher plants, the identification of plant ACs has somewhat remained so slow. In Arabidopsis thaliana alone, only twelve ACs have so far been identified, yet considering the number and diverse nature of processes known to be cAMP-dependent in this plant, these identified ACs are still very much few to account for that. Notably, an additional protein in this plant, termed linker histone-like (AtLHL) protein (encoded by the At3g18035 gene), is annotated to be an AC as result of it containing a putative centre identical to the one commonly found in the other twelve previously confirmed Arabidopsis ACs. In addition, AtLHL is mostly involved in a number of key cellular processes such as heterochromatin formation, DNA repair, apoptosis, embryogenesis, reproduction and disease resistance that are all modulated by cAMP, yet AtLHL still remains unconfirmed as an AC. As a result, we targeted this protein in this study to determine if it is indeed an AC. To begin with, we used computational analysis to assess the 3-dimensional (3D) structure of AtLHL and found that its AC centre is solvent-exposed, amenable to the unhindered access of ATP as a substrate for catalysis. Next, we cloned, partially expressed and affinity purified a truncated version of this protein (AtLHL301−480), followed by assessment of its probable AC activity. Through enzyme immunoassay and mass spectrometry, we showed that the recombinant AtLHL301−480 protein can generate cAMP from ATP in vitro in a manganese-dependent manner that is enhanced by calcium and hydrogen carbonate. In addition, we also showed that the recombinant AtLHL301−480 protein can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli host strain. We then used electrochemistry to evaluate the molecular interaction of AtLHL301−480 with its co-factors and modulators during catalysis and activation, respectively, and found that the protein does this physically. This observation then prompted us to specifically search for the presence (and possibly frequency) of calcium-binding sites within the AtLHL protein. Through in silico analysis and bioinformatic studies, a single binding site in form of a 16-residue calmodulin-binding sequence was predicted. Lastly, we then evaluated the reaction kinetics of AtLHL301−480 and determined that the protein has a Km constant of 0.7 mM and a Vmax constant of 9.2 fmol/min/μg protein. All in all, our study provided adequate evidence in a multi-faceted manner that LHL from A. thaliana is a bona fide AC, whose activity might be involved in control and molecular regulation of the various functions of this protein in this plant.

Background: The study of transient gene expression in cassava plants during virus infection using existing protocols is laborious and may take approximately fifteen weeks due to cassava’s recalcitrance to transformation. The combination of a protoplast system with CRISPR-mediated gene editing promises to shorten the turnaround time from plant tissue culture to high-throughput gene expression screening for candidate genes. Here, we detail a protocol for screening genes associated with the response to South African cassava mosaic virus(SACMV) in cassava protoplasts, with reference to the ubiquitin E3 ligase gene, MeE3L.Methods: Cassava protoplasts of model, and SACMV-susceptible and -tolerant genotypes, were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 hours post-transfection. Relative SACMV DNA accumulation was determined using DpnI-digested total DNA via qPCR, MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using one-way ANOVA, Tukey’s HSD test and the 2-ΔΔCTstatistical method. The MeE3Lexonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations using MAFTT and MEGA-X software. Construction of a phylogenetic tree was done using the Maximum Likelihood method and Jones-Taylor-Thornton (JTT) matrix-based model.Results: The differential expression of unedited and mutant MeE3L during SACMV infectionof model, susceptible and tolerant cassava protoplasts was determined within 7 weeks after commencement of tissue culture. The study also revealed thatSACMV DNA accumulation is cassava genotype-dependent and induces multiple mutations in the tolerant landrace MeE3L homolog. Notably, the susceptible cassava landrace encodes a RINGlessMeE3Lwhich is silenced by SACMV-induced mutations.SACMV also induces mutations which silence the MeE3L RING domain in protoplasts from and tolerant cassava landraces.Conclusions: This protocol presented here halves the turnaround time for high-throughput screening of genes associated with the host response to SACMV. It provides evidence that a cassava E3 ligase is associated with the response to SACMV and forms a basis for validation of these findings by in plantafunctional and interaction studies

BackgroundThe ubiquitylation of proteins is reprogrammed by plant geminiviruses which alter the ubiquitin proteasome system (UPS) to fully infect the host. A RING Finger E3 Ubiquitin Ligase (MeE3L) is located on a major cassava mosaic disease resistance-associated quantitative trait locus. Here, we examine the genetic structure and relative expression of MeE3L (native and gene-edited mutant), and determine how MeE3L affects geminivirus South African cassava mosaic virus (SACMV) DNA accumulation. MethodsCassava protoplasts of model, susceptible and tolerant genotypes were transformed with SACMV infectious clones and/or a CRISPR-editing construct targeting the MeE3L using PEG4000-mediated transfection. DNA and RNA were extracted from transformed protoplasts at 24 hours post-transfection. Relative SACMV load quantitation was determined using DpnI-digested total DNA via qPCR and MeE3L relative expression was determined via reverse transcriptase qPCR, and results were analysed using the 2-ΔΔ method. The MeE3L exonic region was sequenced on the ABI 3500XL Genetic Analyzer platform; and sequences were analysed for mutations and for construction of a phylogenetic tree using the Maximum Likelihood method and Tamura-Nei model.ResultsResults show that SACMV DNA accumulation is cassava genotype-dependent. The study also reveals that native and mutant MeE3L is differentially expressed during SACMV infection in protoplasts of susceptible and tolerant cassava landraces. The susceptible cassava landrace encodes a RINGless MeE3L and the MeE3L base sequence is a determinant of cassava’s response to SACMV. Results further show that SACMV silences the MeE3L RING domain in the susceptible and tolerant landraces; and specifically targets the tolerant MeE3L gene homolog for silencing. ConclusionsThese findings suggest that MeE3L is a target of SACMV, contributing to susceptibility in cassava. The MeE3L base sequence is a determinant of cassava’s response to SACMV. The MeE3L RING domain is actively silenced by SACMV and therefore may be essential for host defence against geminiviruses. The study provides further evidence, in addition to existing literature, that plant E3 ligases are exploited by geminiviruses to enhance pathogenicity.