Pathogen-driven coevolution across the CBP60 plant immune regulator subfamilies confers resilience on the regulator module.

Components of the plant immune signaling network need mechanisms that confer resilience against fast-evolving pathogen effectors that target them. Among eight Arabidopsis CaM-Binding Protein (CBP) 60 family members, AtCBP60g and AtSARD1 are partially functionally redundant, major positive immune regulators, and AtCBP60a is a negative immune regulator. We investigated possible resilience-conferring evolutionary mechanisms among the CBP60a, CBP60g and SARD1 immune regulatory subfamilies. Phylogenetic analysis was used to investigate the times of CBP60 subfamily neofunctionalization. Then, using the pairwise distance rank based on the newly developed analytical platform Protein Evolution Analysis in a Euclidean Space (PEAES), hypotheses of specific coevolutionary mechanisms that could confer resilience on the regulator module were tested. The immune regulator subfamilies diversified around the time of angiosperm divergence and have been evolving very quickly. We detected significant coevolutionary interactions across the immune regulator subfamilies in all of 12 diverse core eudicot species lineages tested. The coevolutionary interactions were consistent with the hypothesized coevolution mechanisms. Despite their unusually fast evolution, members across the CBP60 immune regulator subfamilies have influenced the evolution of each other long after their diversification in a way that could confer resilience on the immune regulator module against fast-evolving pathogen effectors.

The protein turnover of Arabidopsis BPM1 is involved in regulation of flowering time and abiotic stress response.

KEY MESSAGE: Protein degradation is essential in plant growth and development. The stability of Cullin3 substrate adaptor protein BPM1 is regulated by multiple environmental cues pointing on manifold control of targeted protein degradation. A small family of six MATH-BTB genes (BPM1-6) is described in Arabidopsis thaliana. BPM proteins are part of the Cullin E3 ubiquitin ligase complexes and are known to bind at least three families of transcription factors: ERF/AP2 class I, homeobox-leucine zipper and R2R3 MYB. By targeting these transcription factors for ubiquitination and subsequent proteasomal degradation, BPMs play an important role in plant flowering, seed development and abiotic stress response. In this study, we generated BPM1-overexpressing plants that showed an early flowering phenotype, resistance to abscisic acid and tolerance to osmotic stress. We analyzed BPM1-GFP protein stability and found that the protein has a high turnover rate and is degraded by the proteasome 26S in a Cullin-dependent manner. Finally, we found that BPM1 protein stability is environmentally conditioned. Darkness and salt stress triggered BPM1 degradation, whereas elevated temperature enhanced BPM1 stability and accumulation in planta.

The Plastid and Mitochondrial Peptidase Network in Arabidopsis thaliana: A Foundation for Testing Genetic Interactions and Functions in Organellar Proteostasis.

Plant plastids and mitochondria have dynamic proteomes. Protein homeostasis in these organelles is maintained by a proteostasis network containing protein chaperones, peptidases, and their substrate recognition factors. However, many peptidases, as well as their functional connections and substrates, are poorly characterized. This review provides a systematic insight into the organellar peptidase network in Arabidopsis thaliana We present a compendium of known and putative Arabidopsis peptidases and inhibitors, and compare the distribution of plastid and mitochondrial peptidases to the total peptidase complement. This comparison shows striking biases, such as the (near) absence of cysteine and aspartic peptidases and peptidase inhibitors, whereas other peptidase families were exclusively organellar; reasons for such biases are discussed. A genome-wide mRNA-based coexpression data set was generated based on quality controlled and normalized public data, and used to infer additional plastid peptidases and to generate a coexpression network for 97 organellar peptidase baits (1742 genes, making 2544 edges). The graphical network includes 10 modules with specialized/enriched functions, such as mitochondrial protein maturation, thermotolerance, senescence, or enriched subcellular locations such as the thylakoid lumen or chloroplast envelope. The peptidase compendium, including the autophagy and proteosomal systems, and the annotation based on the MEROPS nomenclature of peptidase clans and families, is incorporated into the Plant Proteome Database.

The Plastoglobule-Localized Metallopeptidase PGM48 Is a Positive Regulator of Senescence in Arabidopsis thaliana.

Plastoglobuli (PG) are thylakoid-associated monolayer lipid particles with a specific proteome of ∼30 PG core proteins and isoprenoid and neutral lipids. During senescence, PGs increase in size, reflecting their role in dismantling thylakoid membranes. Here, we show that the only PG-localized peptidase PGM48 positively regulates leaf senescence. We discovered that PGM48 is a member of the M48 peptidase family with PGM48 homologs, forming a clade (M48D) only found in photosynthetic organisms. Unlike the M48A, B, and C clades, members of M48D have no transmembrane domains, consistent with their unique subcellular location in the PG. In vitro assays showed Zn-dependent proteolytic activity and substrate cleavage upstream of hydrophobic residues. Overexpression of PGM48 accelerated natural leaf senescence, whereas suppression delayed senescence. Quantitative proteomics of PG from senescing rosettes of PGM48 overexpression lines showed a dramatically reduced level of CAROTENOID CLEAVAGE ENZYME4 (CCD4) and significantly increased levels of the senescence-induced ABC1 KINASE7 (ABC1K7) and PHYTYL ESTER SYNTHASE1 (PES1). Yeast two-hybrid experiments identified PG core proteins ABC1K3, PES1, and CCD4 as PGM48 interactors, whereas several other PG-localized proteins and chlorophyll degradation enzymes did not interact. We discuss mechanisms through which PGM48 could possibly accelerate the senescence process.

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS.

BACKGROUND: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by" an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called "protospacers". Cascade recognizes invader DNA via short "protospacer adjacent motif" (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as "spacers" by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. RESULTS: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. CONCLUSIONS: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems.

Putative mechanisms of antitumor activity of cyano-substituted heteroaryles in HeLa cells.

Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species.