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.

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.

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.

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.

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.