Jean T. Greenberg

SUMMARY: Phytosulfokine (PSK) is a plant growth‐promoting peptide hormone that is perceived by its cell surface receptors PSKR1 and PSKR2 in Arabidopsis. Plants lacking the PSK receptors show phenotypes consistent with PSK signaling repressing some plant defenses. To gain further insight into the PSK signaling mechanism, comprehensive transcriptional profiling of Arabidopsis treated with PSK was performed, and the effects of PSK treatment on plant defense readouts were monitored. Our study indicates that PSK's major effect is to downregulate defense‐related genes; it has a more modest effect on the induction of growth‐related genes. WRKY transcription factors (TFs) emerged as key regulators of PSK‐responsive genes, sharing commonality with a pathogen‐associated molecular pattern (PAMP) responses, flagellin 22 (flg22), but exhibiting opposite regulatory directions. These PSK‐induced transcriptional changes were accompanied by biochemical and physiological changes that reduced PAMP responses, notably mitogen‐activated protein kinase (MPK) phosphorylation (previously implicated in WRKY activation) and the cell wall modification of callose deposition. Comparison with previous studies using other growth stimuli (the sulfated plant peptide containing sulfated tyrosine [PSY] and Pseudomonas simiae strain WCS417) also reveals WRKY TFs' overrepresentations in these pathways, suggesting a possible shared mechanism involving WRKY TFs for plant growth–defense trade‐off.

Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina 's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N -terminal ER membrane signal anchor region (1-32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system.

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species.

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone’s accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.

Plant plastids generate signals, including some derived from lipids, that need to be mobilized to effect signaling. We used informatics to discover potential plastid membrane proteins involved in microbial responses in Arabidopsis (Arabidopsis thaliana). Among these are proteins co-regulated with the systemic immunity component AZELAIC ACID INDUCED 1, a hybrid proline-rich protein (HyPRP), and HyPRP superfamily members. HyPRPs have a transmembrane domain, a proline-rich region (PRR), and a lipid transfer protein domain. The precise subcellular location(s) and function(s) are unknown for most HyPRP family members. As predicted by informatics, a subset of HyPRPs has a pool of proteins that target plastid outer envelope membranes via a mechanism that requires the PRR. Additionally, two HyPRPs may be associated with thylakoid membranes. Most of the plastid- and nonplastid-localized family members also have pools that localize to the endoplasmic reticulum, plasma membrane, or plasmodesmata. HyPRPs with plastid pools regulate, positively or negatively, systemic immunity against the pathogen Pseudomonas syringae. HyPRPs also regulate the interaction with the plant growth-promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture, and/or biomass. Thus, HyPRPs have broad and distinct roles in immunity, development, and growth responses to microbes and reside at sites that may facilitate signal molecule transport.

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.

Plant plastids generate signals, including some derived from lipids, that need to be mobilized to effect signaling. We used informatics to discover potential plastid membrane proteins involved in microbial responses. Among these are proteins co-regulated with the systemic immunity component AZI1, a hybrid proline-rich protein (HyPRP) and HyPRP superfamily members. HyPRPs have a transmembrane domain, a proline-rich region (PRR) and a lipid transfer protein domain. The precise subcellular location(s) and function(s) is unknown for most HyPRP family members. As predicted by informatics, a subset of HyPRPs have a pool of protein that targets plastid outer envelope membranes (OEMs) via a mechanism that requires the PRR. Additionally, two HyPRPs may be associated with thylakoid membranes. Most of the plastid and non-plastid localized family members also have pools that localize to endoplasmic reticulum, plasma membrane or plasmodesmata. HyPRPs with plastid pools regulate, positively or negatively, systemic immunity against the pathogen Pseudomonas syringae . HyPRPs also regulate the interaction with the plant growth promoting rhizobacteria Pseudomonas simiae WCS417 in the roots to influence colonization, root system architecture and/or biomass. Thus, HyPRPs have broad and distinct roles in immune, development and growth responses to microbes and reside at sites that may facilitate signal molecule transport.

The Arabidopsis plastid-localized ALD1 protein acts in the lysine catabolic pathway that produces infection-induced pipecolic acid (Pip), Pip derivatives, and basal non-Pip metabolite(s). ALD1 is indispensable for disease resistance associated with Pseudomonas syringae infections of naïve plants as well as those previously immunized by a local infection, a phenomenon called systemic acquired resistance (SAR). Pseudomonas syringae is known to associate with mesophyll as well as epidermal cells. To probe the importance of epidermal cells in conferring bacterial disease resistance, we studied plants in which ALD1 was only detectable in the epidermal cells of specific leaves. Local disease resistance and many features of SAR were restored when ALD1 preferentially accumulated in the epidermal plastids at immunization sites. Interestingly, SAR restoration occurred without appreciable accumulation of Pip or known Pip derivatives in secondary distal leaves. Our findings establish that ALD1 has a non-autonomous effect on pathogen growth and defense activation. We propose that ALD1 is sufficient in the epidermis of the immunized leaves to activate SAR, but basal ALD1 and possibly a non-Pip metabolite(s) are also needed at all infection sites to fully suppress bacterial growth. Thus, epidermal plastids that contain ALD1 play a key role in local and whole-plant immune signaling.