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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., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 µL droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morgan, Jelenska, Hensley, Retterer, Morrell-Falvey, Standaert and Greenberg.)

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.

Salicylic acid (SA) is a major defense signal in plants. In Arabidopsis ( Arabidopsis thaliana ), the chloroplast-localized isochorismate pathway is the main source of SA biosynthesis during abiotic stress or pathogen infections. In the first step of the pathway, the enzyme ISOCHORISMATE SYNTHASE1 (ICS1) converts chorismate to isochorismate. An unknown enzyme subsequently converts isochorismate to SA. Here, we show that ICS1 protein levels increase during UV-C stress. To identify proteins that may play roles in SA production by regulating ICS1, we analyzed proteins that coimmunoprecipitated with ICS1 via mass spectrometry. The ICS1 complexes contained a large number of peptides from the PROHIBITIN (PHB) protein family, with PHB3 the most abundant. PHB proteins have diverse biological functions that include acting as scaffolds for protein complex formation and stabilization. PHB3 was reported previously to localize to mitochondria. Using fractionation, protease protection, and live imaging, we show that PHB3 also localizes to chloroplasts, where ICS1 resides. Notably, loss of PHB3 function led to decreased ICS1 protein levels in response to UV-C stress. However, ICS1 transcript levels remain unchanged, indicating that ICS1 is regulated posttranscriptionally. The phb3 mutant displayed reduced levels of SA, the SA-regulated protein PR1, and hypersensitive cell death in response to UV-C and avirulent strains of Pseudomonas syringae and, correspondingly, supported increased growth of P. syringae The expression of a PHB3 transgene in the phb3 mutant complemented all of these phenotypes. We suggest a model in which the formation of PHB3-ICS1 complexes stabilizes ICS1 to promote SA production in response to stress., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Diverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections. Remarkably, entry into the plant cell via endocytosis together with the FLS2 receptor is needed for delivery to vascular tissue and long-distance transport of flg22. This contrasts with known routes of long distance transport of other non-cell-permeant molecules in plants, which require membrane-localized transporters for entry to vascular tissue. Thus, a plasma membrane receptor acts as a transporter to enable access of its ligand to distal trafficking routes., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In plants, cell surface pattern recognition receptors (PRRs) provide a first line of defense against pathogens. Although each PRR recognizes a specific ligand, they share common signaling outputs, such as callose and other cell wall-based defenses. Several PRRs are also important for callose induction in response to the defense signal salicylic acid (SA). The extent to which common components are needed for PRR signaling outputs is not known. The gain-of-function Arabidopsis mutant of ACCELERATED CELL DEATH6 (ACD6) acd6-1 shows constitutive callose production that partially depends on PRRs. ACD6-1 (and ACD6) forms complexes with the PRR FLAGELLIN SENSING2, and ACD6 is needed for responses to several PRR ligands. Thus, ACD6-1 could serve as a probe to identify additional proteins important for PRR-mediated signaling. Candidate signaling proteins (CSPs), identified in our proteomic screen after immunoprecipitation of hemagglutinin (HA)-tagged ACD6-1, include several subfamilies of receptor-like kinase (RLK) proteins and a MECHANO-SENSITIVE CHANNEL OF SMALL CONDUCTANCE-LIKE 4 (MSL4). In acd6-1, CSPs contribute to autoimmunity. In wild type, CSPs are needed for defense against bacteria and callose responses to two or more microbial-derived patterns and an SA agonist. CSPs may function to either i) promote the assembly of signaling complexes, ii) regulate the output of known PRRs, or both.

Effective methods for delivering bioprobes into the cells of intact plants are essential for investigating diverse biological processes. Increasing research on trees, such as Populus spp., for bioenergy applications is driving the need for techniques that work well with tree species. This report introduces vertically aligned carbon nanofiber (VACNF) arrays as a new tool for microdelivery of labeled molecules to Populus leaf tissue and whole plants. We demonstrated that VACNFs penetrate the leaf surface to deliver sub-microliter quantities of solution containing fluorescent or radiolabeled molecules into Populus leaf cells. Importantly, VACNFs proved to be gentler than abrasion with carborundum, a common way to introduce material into leaves. Unlike carborundum, VACNFs did not disrupt cell or tissue integrity, nor did they induce production of hydrogen peroxide, a typical wound response. We show that femtomole to picomole quantities of labeled molecules (fluorescent dyes, small proteins and dextran), ranging from 0.5-500 kDa, can be introduced by VACNFs, and we demonstrate the use of the approach to track delivered probes from their site of introduction on the leaf to distal plant regions. VACNF arrays thus offer an attractive microdelivery method for the introduction of biomolecules and other probes into trees and potentially other types of plants.

Modifications of plant immune complexes by secreted pathogen effectors can trigger strong immune responses mediated by the action of nucleotide binding-leucine-rich repeat immune receptors. Although some strains of the pathogen Pseudomonas syringae harbor effectors that individually can trigger immunity, the plant's response may be suppressed by other virulence factors. This work reveals a robust strategy for immune suppression mediated by HopZ3, an effector in the YopJ family of acetyltransferases. The suppressing HopZ3 effector binds to and can acetylate multiple members of the RPM1 immune complex, as well as two P. syringae effectors that together activate the RPM1 complex. These acetylations modify serine, threonine, lysine, and/or histidine residues in the targets. Through HopZ3-mediated acetylation, it is possible that the whole effector-immune complex is inactivated, leading to increased growth of the pathogen., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA., (© 2014 American Society of Plant Biologists. All rights reserved.)

A central mechanism of virulence of extracellular bacterial pathogens is the injection into host cells of effector proteins that modify host cellular functions. HopW1 is an effector injected by the type III secretion system that increases the growth of the plant pathogen Pseudomonas syringae on the Columbia accession of Arabidopsis. When delivered by P. syringae into plant cells, HopW1 causes a reduction in the filamentous actin (F-actin) network and the inhibition of endocytosis, a known actin-dependent process. When directly produced in plants, HopW1 forms complexes with actin, disrupts the actin cytoskeleton and inhibits endocytosis as well as the trafficking of certain proteins to vacuoles. The C-terminal region of HopW1 can reduce the length of actin filaments and therefore solubilize F-actin in vitro. Thus, HopW1 acts by disrupting the actin cytoskeleton and the cell biological processes that depend on actin, which in turn are needed for restricting P. syringae growth in Arabidopsis.

Cells of infected organisms transport disease defense-related molecules along actin filaments to deliver them to their sites of action to combat the pathogen. To accommodate higher demand for intracellular traffic, plant F-actin density increases transiently during infection or treatment of Arabidopsis with pathogen-associated molecules. Many animal and plant pathogens interfere with actin polymerization and depolymerization to avoid immune responses. Pseudomonas syringae, a plant extracellular pathogen, injects HopW1 effector into host cells to disrupt the actin cytoskeleton and reduce vesicle movement in order to elude defense responses. In some Arabidopsis accessions, however, HopW1 is recognized and causes resistance via an actin-independent mechanism. HopW1 targets isoform 7 of vegetative actin (ACT7) that is regulated by phytohormones and environmental factors. We hypothesize that dynamic changes of ACT7 filaments are involved in plant immunity.

Plant heat shock protein Hsp70 is the major target of HopI1, a virulence effector of pathogenic Pseudomonas syringae. Hsp70 is essential for the virulence function of HopI1. HopI1 directly binds Hsp70 through its C-terminal J domain and stimulates Hsp70 ATP hydrolysis activity in vitro. In plants, HopI1 forms large complexes in association with Hsp70 and induces and recruits cytosolic Hsp70 to chloroplasts, the site of HopI1 localization. Deletion of a central P/Q-rich repeat region disrupts HopI1 virulence but not Hsp70 interactions or association with chloroplasts. Thus, HopI1 must not only bind Hsp70 through its J domain, but likely actively affects Hsp70 activity and/or specificity. At high temperature, HopI1 is dispensable for P. syringae pathogenicity, unless excess Hsp70 is provided. A working hypothesis is that Hsp70 has a defense-promoting activity(s) that HopI1 or high temperature can subvert. Enhanced susceptibility of Hsp70-depleted plants to nonpathogenic strains of P. syringae supports a defense-promoting role for Hsp70.

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.

Plant infection responses result from the interaction of pathogen-derived molecules with host components. For the bacterial pathogen Pseudomonas syringae, these molecules are often effector proteins (Hops) that are injected into plant cells. P. syringae carrying hopW1-1 have restricted host range on some Arabidopsis thaliana accessions. At least two Arabidopsis genomic regions are important for the natural variation that conditions resistance to P. syringae/hopW1-1. HopW1-1 elicits a resistance response, and consequently the accumulation of the signal molecule salicylic acid (SA) and transcripts of HWI1 (HopW1-1-Induced Gene1). This work identified three HopW1-1-interacting (WIN) plant proteins: a putative acetylornithine transaminase (WIN1), a protein phosphatase (WIN2) and a firefly luciferase superfamily protein (WIN3). Importantly, WIN2 and WIN3 are partially required for HopW1-1-induced disease resistance, SA production and HWI1 expression. The requirement for WIN2 is specific for HopW1-1-induced resistance, whereas WIN3 is important for responses to several effectors. Overexpression of WIN2 or WIN3 confers resistance to virulent P. syringae, which is consistent with these proteins being defense components. Several known genes important for SA production or signaling are also partially (EDS1, NIM1/NPR1, ACD6 and ALD1) or strongly (PAD4) required for the robust resistance induced by HopW1-1, suggesting a key role for SA in the HopW1-1-induced resistance response. Finally, WIN1 is an essential protein, the overexpression of which over-rides the resistance response to HopW1-1 (and several other defense-inducing effectors), and delays SA and HWI1 induction. Thus, the WIN proteins have different roles in modulating plant defense.

Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.

Background: The plant pathogen Pseudomonas syringae injects 20-40 different proteins called effectors into host plant cells, yet the functions and sites of action of these effectors in promoting pathogenesis are largely unknown. Plants in turn defend themselves against P. syringae by activating the salicylic acid (SA)-mediated signaling pathway. The P. syringae-specific HopI1 effector has a putative chloroplast-targeting sequence and a J domain. J domains function by activating 70 kDa heat-shock proteins (Hsp70)., Results: HopI1 is a ubiquitous P. syringae virulence effector that acts inside plant cells. When expressed in plants, HopI1 localizes to chloroplasts, the site of SA synthesis. HopI1 causes chloroplast thylakoid structure remodeling and suppresses SA accumulation. HopI1's C terminus has bona fide J domain activity that is necessary for HopI1-mediated virulence and thylakoid remodeling. Furthermore, HopI1-expressing plants have increased heat tolerance, establishing that HopI1 can engage the plant stress-response machinery., Conclusions: These results strongly suggest that chloroplast Hsp70 is targeted by the P. syringae HopI1 effector to promote bacterial virulence by suppressing plant defenses. The targeting of Hsp70 function through J domain proteins is known to occur in a mammalian virus, SV40. However, this is the first example of a bacterial pathogen exploiting a J domain protein to promote pathogenesis through alterations of chloroplast structure and function.

The bacterial plant pathogen Pseudomonas syringae injects a large repertoire of effector proteins into plant cells using a type III secretion apparatus. Effectors can trigger or suppress defences in a host-dependent fashion. Host defences are often accompanied by programmed cell death, while interference with defences is sometimes associated with cell death suppression. We previously predicted the effector repertoire of the sequenced bean pathogen P. syringae pv. syringae (Psy) B728a using bioinformatics. Here we show that PsyB728a is also pathogenic on the model plant species Nicotiana benthamiana (tobacco). We confirm our effector predictions and clone the nearly complete PsyB728a effector repertoire. We find effectors to have different cell death-modulating activities and distinct roles during the infection of the susceptible bean and tobacco hosts. Unexpectedly, we do not find a strict correlation between cell death-eliciting and defence-eliciting activity and between cell death-suppressing activity and defence-interfering activity. Furthermore, we find several effectors with quantitative avirulence activities on their susceptible hosts, but with growth-promoting effects on Arabidopsis thaliana, a species on which PsyB728a does not cause disease. We conclude that P. syringae strains may have evolved large effector repertoires to extend their host ranges or increase their survival on various unrelated plant species.

The plant pathogen Pseudomonas syringae causes disease by secreting a potentially large set of virulence proteins called effectors directly into host cells, their environment, or both, using a type III secretion system (T3SS). Most P. syringae effectors have a common upstream element called the hrp box, and their N-terminal regions have amino acids biases, features that permit their bioinformatic prediction. One of the most prominent biases is a positive serine bias. We previously used the truncated AvrRpt2(81-255) effector containing a serine-rich stretch from amino acids 81 to 100 as a T3SS reporter. Region 81 to 100 of this reporter does not contribute to the secretion or translocation of AvrRpt2 or to putative effector protein chimeras. Rather, the serine-rich region from the N-terminus of AvrRpt2 is important for protein accumulation in bacteria. Most of the N-terminal region (amino acids 15 to 100) is not essential for secretion in culture or delivery to plants. However, portions of this sequence may increase the efficiency of AvrRpt2 secretion, delivery to plants, or both. Two effectors previously identified with the AvrRpt2(81-255) reporter were secreted in culture independently of AvrRpt2, validating the use of the C terminus of AvrRpt2 as a T3SS reporter. Finally, using the reduced AvrRpt2(101-255) reporter, we confirmed seven predicted effectors from P. syringae pv. tomato DC3000, four from P. syringae pv. syringae B728a, and two from P. fluorescens SBW25.

The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isd genes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall-anchored proteins to relay heme-iron across the bacterial envelope.

Expression of cytosolic and plastid acetyl-coenzyme A carboxylase (ACCase) gene families at the mRNA level was analyzed in developing wheat (Triticum aestivum) plants. The major plastid ACCase mRNA level is high in the middle part of the plant and low in roots and leaf blades. An alternative plastid ACCase transcript initiated at a different promoter and using an alternative 5' splice site for the first intron accumulates to its highest level in roots. Cytosolic ACCase mRNA also consists of two species, one of which is present at approximately a constant level, whereas the other accumulates to a high level in the lower sheath section. It is likely that different promoters are also responsible for the two forms of cytosolic ACCase mRNA. The abundances of cytosolic and plastid ACCase mRNAs in the sheath section of the plant are similar. ACCase protein level is significantly lower in the leaf blades, in parallel with changes in the total ACCase mRNA level. Homoeologous ACCase genes show the same expression patterns and similar mRNA levels, suggesting that none of the genes was silenced or acquired new tissue specificity after polyploidization.

Inhibition of growth of the apicomplexan parasite Toxoplasma gondii by aryloxyphenoxypropionate herbicides has been correlated with the inhibition of its acetyl-CoA carboxylase (ACC) by these compounds. Here, full-length and C-terminal fragments of T. gondii apicoplast ACC as well as C-terminal fragments of the cytosolic ACC were expressed in Escherichia coli. The recombinant proteins that were soluble showed the expected enzymatic activities. Yeast gene-replacement strains depending for growth on the expressed T. gondii ACC were derived by complementation of a yeast ACC1 null mutation. In vitro and in vivo tests with aryloxyphenoxypropionates showed that the carboxyltransferase domain of the apicoplast T. gondii ACC is the target for this class of inhibitors. The cytosolic T. gondii ACC is resistant to aryloxyphenoxypropionates. Both T. gondii isozymes are resistant to cyclohexanediones, another class of inhibitors targeting the ACC of grass plastids.