Pathogen effectors: What do they do at plasmodesmata?

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.

Aluminum toxicity and aluminum stress-induced physiological tolerance responses in higher plants.

Aluminum (Al) precipitates in acidic soils having a pH < 5.5, in the form of conjugated organic and inorganic ions. Al-containing minerals solubilized in the soil solution cause several negative impacts in plants when taken up along with other nutrients. Moreover, a micromolar concentration of Al present in the soil is enough to induce several irreversible toxicity symptoms such as the rapid and transient over-generation of reactive oxygen species (ROS) such as superoxide anion (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), resulting in oxidative bursts. In addition, significant reductions in water and nutrient uptake occur which imposes severe stress in the plants. However, some plants have developed Al-tolerance by stimulating the secretion of organic acids like citrate, malate, and oxalate, from plant roots. Genes responsible for encoding such organic acids, play a critical role in Al tolerance. Several transporters involved in Al resistance mechanisms are members of the Aluminum-activated Malate Transporter (ALMT), Multidrug and Toxic compound Extrusion (MATE), ATP-Binding Cassette (ABC), Natural resistance-associated macrophage protein (Nramp), and aquaporin gene families. Therefore, in the present review, the discussion of the global extension and probable cause of Al in the environment and mechanisms of Al toxicity in plants are followed by detailed emphasis on tolerance mechanisms. We have also identified and categorized the important transporters that secrete organic acids and outlined their role in Al stress tolerance mechanisms in crop plants. The information provided here will be helpful for efficient exploration of the available knowledge to develop Al tolerant crop varieties.

The Conserved Arginine Required for AvrRps4 Processing Is Also Required for Recognition of Its N-Terminal Fragment in Lettuce.

Pathogens utilize a repertoire of effectors to facilitate pathogenesis, but when the host recognizes one of them, it causes effector-triggered immunity. The Pseudomonas type III effector AvrRps4 is a bipartite effector that is processed in planta into a functional 133-amino acid N-terminus (AvrRps4-N) and 88-amino acid C-terminus (AvrRps4-C). Previous studies found AvrRps4-C to be sufficient to trigger the hypersensitive response (HR) in turnip. In contrast, our recent work found that AvrRps4-N but not AvrRps4-C triggered HR in lettuce, whereas both were required for resistance induction in Arabidopsis. Here, we initially compared AvrRps4 recognition by turnip and lettuce using transient expression. By serial truncation, we identified the central conserved region consisting of 37 amino acids as essential for AvrRps4-N recognition, whereas the putative type III secretion signal peptide or the C-terminal 13 amino acids were dispensable. Surprisingly, the conserved arginine at position 112 (R112) that is required for full-length AvrRps4 processing is also required for the recognition of AvrRps4-N by lettuce. Mutating R112 to hydrophobic leucine or negatively charged glutamate abolished the HR-inducing capacity of AvrRps4-N, while a positively charged lysine at this position resulted in a slow and weak HR. Together, our results suggest an AvrRps4-N recognition-specific role of R112 in lettuce.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Generating Transgenic Arabidopsis Plants for Functional Analysis of Pathogen Effectors and Corresponding R Proteins.

Inducible expression of a pathogen effector has been proven to be a powerful strategy for dissecting its virulence and avirulence functions. However, leaky expression of some effector proteins can cause drastic physiological changes, such as growth retardation, accelerated senescence, and sterility. Unfortunately, leaky expression from current inducible vectors is unavoidable. To overcome these problems, a highly efficient Arabidopsis transformation protocol is described here, which allows the generation of hundreds to over a thousand T1 plants for selecting appropriate lines. In addition, since transgenic silencing is frequently observed, a principle for screening stable transgenic plants is also introduced.

Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporters.

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background-free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL-LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non-plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters.

Copper (Cu) is an essential plant micronutrient. Under scarcity, Cu2+ is reduced to Cu+ and taken up through specific high-affinity transporters (COPTs). In Arabidopsis, the COPT family consists of six members, either located at the plasma membrane (COPT1, COPT2, and COPT6) or in internal membranes (COPT3 and COPT5). Cu uptake by COPT proteins has been mainly assessed through complementation studies in corresponding yeast mutants, but the mechanism of this transport has not been elucidated. To test whether Cu is incorporated by an electrogenic mechanism, electrophysiological changes induced by Cu addition were studied in Arabidopsis thaliana. Mutant (T-DNA insertion mutants, copt2-1 and copt5-2) and overexpressing lines (COPT1OE and COPT5OE) with altered expression of COPT transporters were compared to wild-type plants. No significant changes of the membrane potential (Em) were detected, regardless of genotype or Cu concentration supplied. In contrast, membrane depolarization was detected in response to iron supply in both wild-type and in mutant or transgenic plants. Similar results were obtained for trans-plant potentials (TPP). GFP fusions of the plasma membrane COPT2 and the internal COPT5 transporters were expressed in Xenopus laevis oocytes to potentiate Cu uptake signals, and the cRNA-injected oocytes were tested for electrical currents upon Cu addition using two-electrode voltage clamp. Results with oocytes confirmed those obtained in plants. Cu accumulation in injected oocytes was measured by ICP-OES, and a significant increase in Cu content with respect to controls occurred in oocytes expressing COPT2:GFP. The possible mechanisms driving this transport are discussed in this manuscript.

Soybean TIP Gene Family Analysis and Characterization of GmTIP1;5 and GmTIP2;5 Water Transport Activity.

Soybean, one of the most important crops worldwide, is severely affected by abiotic stress. Drought and flooding are the major abiotic stresses impacting soybean yield. In this regard, understanding water uptake by plants, its utilization and transport has great importance. In plants, water transport is mainly governed by channel forming aquaporin proteins (AQPs). Tonoplast intrinsic proteins (TIPs) belong to the plant-specific AQP subfamily and are known to have a role in abiotic stress tolerance. In this study, 23 soybean TIP genes were identified based on the latest soybean genome annotation. TIPs were characterized based on conserved structural features and phylogenetic distribution. Expression analysis of soybean TIP genes in various tissues and under abiotic stress conditions demonstrated tissue/stress-response specific differential expression. The natural variations for TIP genes were analyzed using whole genome re-sequencing data available for a set of 106 diverse soybean genotypes including wild types, landraces and elite lines. Results revealed 81 single-nucleotide polymorphisms (SNPs) and several large insertions/deletions in the coding region of TIPs. Among these, non-synonymous SNPs are most likely to have a greater impact on protein function and are candidates for molecular studies as well as for the development of functional markers to assist breeding. The solute transport function of two TIPs was further validated by expression in Xenopus laevis oocytes. GmTIP1;5 was shown to facilitate the rapid movement of water across the oocyte membrane, while GmTIP2;5 facilitated the movement of water and boric acid. The present study provides an initial insight into the possible roles of soybean TIP genes under abiotic stress conditions. Our results will facilitate elucidation of their precise functions during abiotic stress responses and plant development, and will provide potential breeding targets for modifying water movement in soybean.

The Arabidopsis immune regulator SRFR1 dampens defences against herbivory by Spodoptera exigua and parasitism by Heterodera schachtii.

Plants have developed diverse mechanisms to fine tune defence responses to different types of enemy. Cross-regulation between signalling pathways may allow the prioritization of one response over another. Previously, we identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)-dependent effector-triggered immunity against the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4. The use of multiple stresses is a powerful tool to further define gene function. Here, we examined whether SRFR1 also impacts resistance to a herbivorous insect in leaves and to a cyst nematode in roots. Interestingly, srfr1-1 plants showed increased resistance to herbivory by the beet army worm Spodoptera exigua and to parasitism by the cyst nematode Heterodera schachtii compared with the corresponding wild-type Arabidopsis accession RLD. Using quantitative real-time PCR (qRT-PCR) to measure the transcript levels of salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathway genes, we found that enhanced resistance of srfr1-1 plants to S. exigua correlated with specific upregulation of the MYC2 branch of the JA pathway concurrent with suppression of the SA pathway. In contrast, the greater susceptibility of RLD was accompanied by simultaneously increased transcript levels of SA, JA and JA/ET signalling pathway genes. Surprisingly, mutation of either SRFR1 or EDS1 increased resistance to H. schachtii, indicating that the concurrent presence of both wild-type genes promotes susceptibility. This finding suggests a novel form of resistance in Arabidopsis to the biotrophic pathogen H. schachtii or a root-specific regulation of the SA pathway by EDS1, and places SRFR1 at an intersection between multiple defence pathways.

Functions of EDS1-like and PAD4 genes in grapevine defenses against powdery mildew.

The molecular interactions between grapevine and the obligate biotrophic fungus Erysiphe necator are not understood in depth. One reason for this is the recalcitrance of grapevine to genetic modifications. Using defense-related Arabidopsis mutants that are susceptible to pathogens, we were able to analyze key components in grapevine defense responses. We have examined the functions of defense genes associated with the salicylic acid (SA) pathway, including ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), EDS1-LIKE 2 (EDL2), EDL5 and PHYTOALEXIN DEFICIENT 4 (PAD4) of two grapevine species, Vitis vinifera cv. Cabernet Sauvignon, which is susceptible to E. necator, and V. aestivalis cv. Norton, which is resistant. Both VaEDS1 and VvEDS1 were previously found to functionally complement the Arabidopsis eds1-1 mutant. Here we show that the promoters of both VaEDS1 and VvEDS1 were induced by SA, indicating that the heightened defense of Norton is related to its high SA level. Other than Va/VvEDS1, only VaEDL2 complemented Arabidopsis eds1-1, whereas Va/VvPAD4 did not complement Arabidopsis pad4-1. Bimolecular fluorescence complementation results indicated that Vitis EDS1 and EDL2 proteins interact with Vitis PAD4 and AtPAD4, suggesting that Vitis EDS1/EDL2 forms a complex with PAD4 to confer resistance, as is known from Arabidopsis. However, Vitis EDL5 and PAD4 did not interact with Arabidopsis EDS1 or PAD4, correlating with their inability to function in Arabidopsis. Together, our study suggests a more complicated EDS1/PAD4 module in grapevine and provides insight into molecular mechanisms that determine disease resistance levels in Vitis species native to the North American continent.

Transport of boron by the tassel-less1 aquaporin is critical for vegetative and reproductive development in maize.

The element boron (B) is an essential plant micronutrient, and B deficiency results in significant crop losses worldwide. The maize (Zea mays) tassel-less1 (tls1) mutant has defects in vegetative and inflorescence development, comparable to the effects of B deficiency. Positional cloning revealed that tls1 encodes a protein in the aquaporin family co-orthologous to known B channel proteins in other species. Transport assays show that the TLS1 protein facilitates the movement of B and water into Xenopus laevis oocytes. B content is reduced in tls1 mutants, and application of B rescues the mutant phenotype, indicating that the TLS1 protein facilitates the movement of B in planta. B is required to cross-link the pectic polysaccharide rhamnogalacturonan II (RG-II) in the cell wall, and the percentage of RG-II dimers is reduced in tls1 inflorescences, indicating that the defects may result from altered cell wall properties. Plants heterozygous for both tls1 and rotten ear (rte), the proposed B efflux transporter, exhibit a dosage-dependent defect in inflorescence development under B-limited conditions, indicating that both TLS1 and RTE function in the same biological processes. Together, our data provide evidence that TLS1 is a B transport facilitator in maize, highlighting the importance of B homeostasis in meristem function.

Members of the NPF3 transporter subfamily encode pathogen-inducible nitrate/nitrite transporters in grapevine and Arabidopsis.

Vitis vinifera, the major grapevine species cultivated for wine production, is very susceptible to Erysiphe necator, the causal agent of powdery mildew (PM). This obligate biotrophic fungal pathogen attacks both leaf and berry, greatly affecting yield and quality. To investigate possible mechanisms of nutrient acquisition by successful biotrophs, we characterized a candidate NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER FAMILY (NPF, formerly NRT1/PTR) member, grapevine NFP3.2, that was up-regulated in E. necator-inoculated susceptible V. vinifera Cabernet Sauvignon leaves, but not in resistant V. aestivalis Norton. Expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements showed that VvNPF3.2 is a low-affinity transporter for both nitrate and nitrite and displays characteristics of NPF members from other plants. We also cloned the Arabidopsis ortholog, AtNPF3.1, and showed that AtNPF3.1 similarly transported nitrate and nitrite with low affinity. With an Arabidopsis triple mutant that is susceptible to E. necator, we found that AtNPF3.1 is up-regulated in the leaves of infected Arabidopsis similarly to VvNPF3.2 in susceptible grapevine leaves. Expression of the reporter ß-glucuronidase (GUS) driven by the promoter of VvNPF3.2 or AtNPF3.1 in Arabidopsis indicated that both transporters are expressed in vascular tissue, with expression in major and minor veins, respectively. Interestingly, the promoter of VvNPF3.2 allowed induced expression of GUS in minor veins in PM-infected leaves. Our experiments lay the groundwork for investigating the manipulation of host nutrient distribution by biotrophic pathogens and characterizing physiological variables in the pathogenesis of this difficult to study grapevine disease.

The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance.

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.

Arabidopsis OPT6 is an oligopeptide transporter with exceptionally broad substrate specificity.

Oligopeptide transporters (OPTs) are found in fungi, bacteria and plants. The nine Arabidopsis thaliana OPT genes are expressed mainly in the vasculature and are thought to transport tetra- and pentapeptides, and peptide-like substrates such as glutathione. Expression of AtOPT6 in Xenopus laevis oocytes demonstrated that AtOPT6 transports many tetra- and pentapeptides. In addition, AtOPT6 transported reduced glutathione (GSH), a tripeptide, but not oxidized glutathione (GSSG). Recent data showed that Candida albicans OPTs can transport peptides up to eight amino acids in length. AtOPT6 transported mammalian signaling peptides up to 10 amino acids in length and, in addition, known plant development- and nematode pathogenesis-associated peptides up to 13 amino acids long. AtOPT6 displayed high affinity for penta- and dodecapeptides, but low affinity for GSH. In comparison the Saccharomyces cerevisiae ScOPT1 was incapable of transporting any of the longer peptides tested. These data demonstrate the necessity of experimentally determining substrate specificity of individual OPTs, and lay a foundation for structure/function studies. Characterization of the AtOPT6 substrate range provides a basis for investigating the possible physiological function of AtOPT6 in peptide signaling and thiol transport in response to stress.

Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade.

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.

Electrophysiological characterization of the Arabidopsis avrRpt2-specific hypersensitive response in the absence of other bacterial signals.

The hypersensitive response (HR) is defined as rapid cell collapse at the infection site and often accompanies plant resistance. The physiological processes leading to HR are not well understood. Here, we report an electrophysiological characterization of bacterial HR caused by a single avirulence gene in the absence of other bacterial signals. We used dexamethasone (dex)-inducible transgenic Arabidopsis (Arabidopsis thaliana) plants containing the avrRpt2 gene from Pseudomonas syringae pv tomato. Membrane depolarization in these plants began 1 to 1.5 h after dex application, hours before electrolyte leakage. Progressive depolarization was a sensitive early indicator of HR that occurred only in Arabidopsis leaf cells expressing both avrRpt2 and a functional RPS2 gene. Hyperpolarization of fully depolarized membranes by fusicoccin, a fungal toxin that activates the H(+)-ATPase, indicates that depolarization did not result from a nonfunctional pump or leaky membranes. Depolarization and electrolyte leakage were inhibited in RPS2 plants by the calcium channel blocker LaCl(3), highly correlating these events and suggesting that Ca(2+) entry into cells is required for both. Also correlated were inhibition of depolarization, electrolyte leakage, and HR following salicylic acid pretreatment. In salicylic acid-pretreated RPS2 seedlings, avrRpt2 transcript was produced after dex treatment. However, AvrRpt2 protein accumulation was greatly reduced, suggesting a possible mechanism for inhibition of HR in plants with induced resistance. This experimental system is a very sensitive assay that lends itself to the dissection of physiological processes leading to HR in plants, and provides a baseline for future research within a genetic framework.

Electrical potentials of plant cell walls in response to the ionic environment.

Electrical potentials in cell walls (psi(Wall)) and at plasma membrane surfaces (psi(PM)) are determinants of ion activities in these phases. The psi(PM) plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of psi(Wall). psi(Wall) from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of psi(Wall) with effectiveness in the order Al(3+) > La(3+) > H(+) > Cu(2+) > Ni(2+) > Ca(2+) > Co(2+) > Cd(2+) > Mg(2+) > Zn(2+) > hexamethonium(2+) > Rb(+) > K(+) > Cs(+) > Na(+). This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of psi(Wall), and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for psi(Wall) were in close agreement, usually, and we consider psi(Wall) to be at least proportional to the actual Donnan potentials. psi(Wall) and psi(PM) display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. psi(Wall) is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor.

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.