Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

The receptor-like cytoplasmic kinase CDG1 negatively regulates Arabidopsis pattern-triggered immunity and is involved in AvrRpm1-induced RIN4 phosphorylation.

Arabidopsis CDG1 negatively regulates flg22- and chitin-triggered immunity by promoting FLS2 and CERK1 degradation and is partially required for bacterial effector AvrRpm1-induced RIN4 phosphorylation. Negative regulators play indispensable roles in pattern-triggered immunity in plants by preventing sustained immunity impeding growth. Here, we report Arabidopsis thaliana CONSTITUTIVE DIFFERENTIAL GROWTH1 (CDG1), a receptor-like cytoplasmic kinase VII member, as a negative regulator of bacterial flagellin/flg22- and fungal chitin-triggered immunity. CDG1 can interact with the flg22 receptor FLAGELLIN SENSITIVE2 (FLS2) and chitin co-receptor CHITIN ELICITOR RECEPTOR KINASE1 (CERK1). CDG1 overexpression impairs flg22 and chitin responses by promoting the degradation of FLS2 and CERK1. This process requires the kinase activity of MEK KINASE1 (MEKK1), but not the Plant U-Box (PUB) ubiquitin E3 ligases PUB12 and PUB13. Interestingly, the Pseudomonas syringae effector AvrRpm1 can induce CDG1 to interact with its host target RPM1-INTERACTING PROTEIN4 (RIN4), which depends on the ADP-ribosyl transferase activity of AvrRpm1. CDG1 is capable of phosphorylating RIN4 in vitro at multiple sites including Thr166 and the AvrRpm1-induced Thr166 phosphorylation of RIN4 is diminished in cdg1 null plants. Accordingly, CDG1 knockout attenuates AvrRpm1-induced hypersensitive response and increases the growth of AvrRpm1-secreting bacteria in plants. Unexpectedly, AvrRpm1 can also induce FLS2 depletion, which is fully dependent on RIN4 and partially dependent on CDG1, but does not require the kinase activity of MEKK1. Collectively, this study reveals previously unknown functions of CDG1 in both pattern-triggered immunity and effector-triggered susceptibility in plants.

Plant Sense: The Rise of Calcium Channels.

Although calcium (Ca2+) elevation triggered by abiotic and biotic stimuli has long been a documented phenomenon in plants, the mechanism underlying the control of Ca2+ spikes remains elusive. Recent progress, reported by Tian et al., Wang et al., Yu et al., Jiang et al., and Wu et al., has been made in elucidating how Ca2+ channels are controlled during pathogen attack, cell death, and salt or hydrogen peroxide sensing.

Dynamic G protein alpha signaling in Arabidopsis innate immunity.

Heterotrimeric G proteins composed of Gα, Gß and Gγ subunits are evolutionarily conserved signaling modules involved in diverse biological processes in plants and animals. The role and action of Gα remain largely enigmatic in plant innate immunity. We have recently demonstrated that Arabidopsis Gα (GPA1) is a key component of a new immune signaling pathway activated by bacteria-secreted proteases. Here we show that GPA1 is also involved in the signaling network of Arabidopsis in response to the bacterial flagellin epitope flg22. Specifically, GPA1 plays a pivotal role in an immune pathway involving the flg22 receptor FLS2, co-receptor BAK1, Regulator of G Signaling 1 (RGS1), and Arabidopsis Gß (AGB1), in which flg22 elicits GPA1/AGB1 dissociation from the FLS2/BAK1/RGS1 receptor complex. Consequently, we observed flg22-induced degradation of FLS2, BAK1 and RGS1 but not GPA1 or AGB1. We also found that GPA1 constitutively interacts with the NADPH oxidase RbohD to potentiate flg22-induced ROS burst independently of the central cytoplasmic kinase BIK1. Taken together, our work sheds multiple novel insights into the functions and regulatory mechanisms of GPA1 in Arabidopsis innate immunity.

Engineering Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen.

Artificial microRNA (amiRNA) technology offers reversible and flexible gene inactivation and complements genome-editing technologies. However, obtaining transgenic plants with maximal gene silencing remains a major technical challenge in current amiRNA applications. Here, we incorporated an empirically determined feature of effective amiRNAs to the amiRNA design and in silico generated a database containing 533,429 gene-specific amiRNAs for silencing 27,136 genes in Arabidopsis (Arabidopsis thaliana), with a genome coverage of 98.87%. In both single-gene and multiple-gene silencing, we observed an overall improvement in performance by amiRNAs designed using our strategy in Arabidopsis protoplasts and transgenic plants. In addition, the endogenous tRNA-processing system was used to generate multiple amiRNAs from tRNA-pre-amiRNA tandem repeats for multiplex gene silencing. An intronic amiRNA-producing fluorescent reporter was explored as a visual screening strategy for transgenic Arabidopsis and rice (Oryza sativa) plants with maximal whole-plant or cell type-specific gene silencing. These improvements enable the amiRNA technology to be a functional gene knockout tool for basic and applied plant research.