GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance.

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata.

Molecular characterization and expression analysis of the Na+/H+ exchanger gene family in Medicago truncatula.

One important mechanism plants use to cope with salinity is keeping the cytosolic Na+ concentration low by sequestering Na+ in vacuoles, a process facilitated by Na+/H+ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~ 20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl- contents increased by 152 and 162%, respectively. Na+ exclusion may be responsible for the relatively smaller increase in Na+ concentration in roots under salt stress as compared to Cl-. Decline in tissue K+ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na+ and K + . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na+ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis.

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H(2)O(2) accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response-causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H(2)O(2) during both gene-for-gene and nonhost resistance responses.

IAA-producing plant growth promoting rhizobacteria from Ceanothus velutinus enhance cutting propagation efficiency and Arabidopsis biomass.

Climate-induced drought impacts plant growth and development. Recurring droughts increase the demand for water for food production and landscaping. Native plants in the Intermountain West region of the US are of keen interest in low water use landscaping as they are acclimatized to dry and cold environments. These native plants do very well at their native locations but are difficult to propagate in landscape. One of the possible reasons is the lack of associated microbiome in the landscaping. Microbiome in the soil contributes to soil health and impacts plant growth and development. Here, we used the bulk soil from the native plant Ceanothus velutinus (snowbrush ceanothus) as inoculant to enhance its propagation. Snowbrush ceanothus is an ornamental plant for low-water landscaping that is hard to propagate asexually. Using 50% native bulk soil as inoculant in the potting mix significantly improved the survival rate of the cuttings compared to no-treated cuttings. Twenty-four plant growth-promoting rhizobacteria (PGPR) producing indole acetic acid (IAA) were isolated from the rhizosphere and roots of the survived snowbrush. Seventeen isolates had more than 10µg/mL of IAA were shortlisted and tested for seven different plant growth-promoting (PGP) traits; 76% showed nitrogen-fixing ability on Norris Glucose Nitrogen free media,70% showed phosphate solubilization activity, 76% showed siderophore production, 36% showed protease activity, 94% showed ACC deaminase activity on DF-ACC media, 76% produced catalase and all of isolates produced ammonia. Eight of seventeen isolates, CK-6, CK-22, CK-41, CK-44, CK-47, CK-50, CK-53, and CK-55, showed an increase in shoot biomass in Arabidopsis thaliana. Seven out of eight isolates were identified as Pseudomonas, except CK-55, identified as Sphingobium based on 16S rRNA gene sequencing. The shortlisted isolates are being tested on different grain and vegetable crops to mitigate drought stress and promote plant growth.

Strategies for combating plant salinity stress: the potential of plant growth-promoting microorganisms.

Global climate change and the decreasing availability of high-quality water lead to an increase in the salinization of agricultural lands. This rising salinity represents a significant abiotic stressor that detrimentally influences plant physiology and gene expression. Consequently, critical processes such as seed germination, growth, development, and yield are adversely affected. Salinity severely impacts crop yields, given that many crop plants are sensitive to salt stress. Plant growth-promoting microorganisms (PGPMs) in the rhizosphere or the rhizoplane of plants are considered the "second genome" of plants as they contribute significantly to improving the plant growth and fitness of plants under normal conditions and when plants are under stress such as salinity. PGPMs are crucial in assisting plants to navigate the harsh conditions imposed by salt stress. By enhancing water and nutrient absorption, which is often hampered by high salinity, these microorganisms significantly improve plant resilience. They bolster the plant's defenses by increasing the production of osmoprotectants and antioxidants, mitigating salt-induced damage. Furthermore, PGPMs supply growth-promoting hormones like auxins and gibberellins and reduce levels of the stress hormone ethylene, fostering healthier plant growth. Importantly, they activate genes responsible for maintaining ion balance, a vital aspect of plant survival in saline environments. This review underscores the multifaceted roles of PGPMs in supporting plant life under salt stress, highlighting their value for agriculture in salt-affected areas and their potential impact on global food security.

Drought stress acclimation imparts tolerance to Sclerotinia sclerotiorum and Pseudomonas syringae in Nicotiana benthamiana.

Acclimation of plants with an abiotic stress can impart tolerance to some biotic stresses. Such a priming response has not been widely studied. In particular, little is known about enhanced defense capacity of drought stress acclimated plants to fungal and bacterial pathogens. Here we show that prior drought acclimation in Nicotiana benthamiana plants imparts tolerance to necrotrophic fungus, Sclerotinia sclerotiorum, and also to hemi-biotrophic bacterial pathogen, Pseudomonas syringae pv. tabaci. S. sclerotiorum inoculation on N. benthamiana plants acclimated with drought stress lead to less disease-induced cell death compared to non-acclimated plants. Furthermore, inoculation of P. syringae pv. tabaci on N. benthamiana plants acclimated to moderate drought stress showed reduced disease symptoms. The levels of reactive oxygen species (ROS) in drought acclimated plants were highly correlated with disease resistance. Further, in planta growth of GFPuv expressing P. syringae pv. tabaci on plants pre-treated with methyl viologen showed complete inhibition of bacterial growth. Taken together, these experimental results suggested a role for ROS generated during drought acclimation in imparting tolerance against S. sclerotiorum and P. syringae pv. tabaci. We speculate that the generation of ROS during drought acclimation primed a defense response in plants that subsequently caused the tolerance against the pathogens tested.

Salinity stress tolerance prediction for biomass-related traits in maize (Zea mays L.) using genome-wide markers.

Maize (Zea mays L.) is the third most important cereal crop after rice (Oryza sativa) and wheat (Triticum aestivum). Salinity stress significantly affects vegetative biomass and grain yield and, therefore, reduces the food and silage productivity of maize. Selecting salt-tolerant genotypes is a cumbersome and time-consuming process that requires meticulous phenotyping. To predict salt tolerance in maize, we estimated breeding values for four biomass-related traits, including shoot length, shoot weight, root length, and root weight under salt-stressed and controlled conditions. A five-fold cross-validation method was used to select the best model among genomic best linear unbiased prediction (GBLUP), ridge-regression BLUP (rrBLUP), extended GBLUP, Bayesian Lasso, Bayesian ridge regression, BayesA, BayesB, and BayesC. Examination of the effect of different marker densities on prediction accuracy revealed that a set of low-density single nucleotide polymorphisms obtained through filtering based on a combination of analysis of variance and linkage disequilibrium provided the best prediction accuracy for all the traits. The average prediction accuracy in cross-validations ranged from 0.46 to 0.77 across the four derived traits. The GBLUP, rrBLUP, and all Bayesian models except BayesB demonstrated comparable levels of prediction accuracy that were superior to the other modeling approaches. These findings provide a roadmap for the deployment and optimization of genomic selection in breeding for salt tolerance in maize.

Exploration of the rhizosphere microbiome of native plant Ceanothus velutinus - an excellent resource of plant growth-promoting bacteria.

Continuous demand for an increase in food production due to climate change and a steady rise in world population requires stress-resilient, sustainable agriculture. Overuse of chemical fertilizers and monoculture farming to achieve this goal deteriorated soil health and negatively affected its microbiome. The rhizosphere microbiome of a plant plays a significant role in its growth and development and promotes the plant's overall health through nutrient uptake/availability, stress tolerance, and biocontrol activity. The Intermountain West (IW) region of the US is rich in native plants recommended for low water use landscaping because of their drought tolerance. The rhizosphere microbiome of these native plants is an excellent resource for plant growth-promoting rhizobacteria (PGPR) to use these microbes as biofertilizers and biostimulants to enhance food production, mitigate environmental stresses and an alternative for chemical fertilizer, and improve soil health. Here, we isolated, purified, identified, and characterized 64 bacterial isolates from a native plant, Ceanothus velutinus, commonly known as snowbrush ceanothus, from the natural habitat and the greenhouse-grown native soil-treated snowbrush ceanothus plants. We also conducted a microbial diversity analysis of the rhizosphere of greenhouse-grown native soil-treated and untreated plants (control). Twenty-seven of the 64 isolates were from the rhizosphere of the native region, and 36 were from the greenhouse-grown native soil-treated plants. These isolates were also tested for plant growth-promoting (PGP) traits such as their ability to produce catalase, siderophore, and indole acetic acid, fix atmospheric nitrogen and solubilize phosphate. Thirteen bacterial isolates tested positive for all five plant growth-promoting abilities and belonged to the genera Pantoea, Pseudomonas, Bacillus, and Ancylobacter. Besides, there are isolates belonging to the genus Streptomyces, Bacillus, Peribacillus, Variovorax, Xenophilus, Brevundimonas, and Priestia, which exhibit at least one of the plant growth-promoting activities. This initial screen provided a list of potential PGPR to test for plant health improvement on model and crop plants. Most of the bacterial isolates in this study have a great potential to become biofertilizers and bio-stimulants.

Morphological, physiological, biochemical, and transcriptome studies reveal the importance of transporters and stress signaling pathways during salinity stress in Prunus.

The almond crop has high economic importance on a global scale, but its sensitivity to salinity stress can cause severe yield losses. Salt-tolerant rootstocks are vital for crop economic feasibility under saline conditions. Two commercial rootstocks submitted to salinity, and evaluated through different parameters, had contrasting results with the survival rates of 90.6% for 'Rootpac 40' (tolerant) and 38.9% for 'Nemaguard' (sensitive) under salinity (Electrical conductivity of water = 3 dS m-1). Under salinity, 'Rootpac 40' accumulated less Na and Cl and more K in leaves than 'Nemaguard'. Increased proline accumulation in 'Nemaguard' indicated that it was highly stressed by salinity compared to 'Rootpac 40'. RNA-Seq analysis revealed that a higher degree of differential gene expression was controlled by genotype rather than by treatment. Differentially expressed genes (DEGs) provided insight into the regulation of salinity tolerance in Prunus. DEGs associated with stress signaling pathways and transporters may play essential roles in the salinity tolerance of Prunus. Some additional vital players involved in salinity stress in Prunus include CBL10, AKT1, KUP8, Prupe.3G053200 (chloride channel), and Prupe.7G202700 (mechanosensitive ion channel). Genetic components of salinity stress identified in this study may be explored to develop new rootstocks suitable for salinity-affected regions.

Functional role of formate dehydrogenase 1 (FDH1) for host and nonhost disease resistance against bacterial pathogens.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.

Genome-wide identification and expression profiling of glutathione S-transferase family under multiple abiotic and biotic stresses in Medicago truncatula L.

Glutathione transferases (GSTs) constitute an ancient, ubiquitous, multi-functional antioxidant enzyme superfamily that has great importance on cellular detoxification against abiotic and biotic stresses as well as plant development and growth. The present study aimed to a comprehensive genome-wide identification and functional characterization of GST family in one of the economically important legume plants-Medicago truncatula. Here, we have identified a total of ninety-two putative MtGST genes that code for 120 proteins. All these members were classified into twelve classes based on their phylogenetic relationship and the presence of structural conserved domain/motif. Among them, 7 MtGST gene pairs were identified to have segmental duplication. Expression profiling of MtGST transcripts revealed their high level of organ/tissue-specific expression in most of the developmental stages and anatomical tissues. The transcripts of MtGSTU5, MtGSTU8, MtGSTU17, MtGSTU46, and MtGSTU47 showed significant up-regulation in response to various abiotic and biotic stresses. Moreover, transcripts of MtGSTU8, MtGSTU14, MtGSTU28, MtGSTU30, MtGSTU34, MtGSTU46 and MtGSTF8 were found to be highly upregulated in response to drought treatment for 24h and 48h. Among the highly stress-responsive MtGST members, MtGSTU17 showed strong affinity towards its conventional substrates reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) with the lowest binding energy of-5.7 kcal/mol and -6.5 kcal/mol, respectively. Furthermore, the substrate-binding site residues of MtGSTU17 were found to be highly conserved. These findings will facilitate the further functional and evolutionary characterization of GST genes in Medicago.

The ATPase Activity of Escherichia coli Expressed AAA+-ATPase Protein.

ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA+-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In general, the substrate 2-amino-6-mercapto-7-methylpurine riboside (methylthioguanosine, a guanosine analog: MESG) is enzymatically converted in the presence of Pi by purine nucleoside phosphorylase (PNP) to ribose 1-phosphate and 2-amino-6-mercapto-7-methylpurine. The spectrophotometric shift in maximum absorbance at 330 nm for the MESG substrate and subsequent conversion product at 360 nm due to enzymatic conversion was measured. The GCN4-His-tagged recombinant protein was expressed in Escherichia coli BL21 cells and purified using Ni-NTA column. This purified protein was then used for the quantitation of Pi in solution or the continuous determination of Pi released due to the ATPase activity of GCN4, an AAA+-ATPase protein conserved in many eukaryotes, which in plants regulates stomatal aperture during biotic and abiotic stress in plants.

Linking diverse salinity responses of 14 almond rootstocks with physiological, biochemical, and genetic determinants.

Fourteen commercial almond rootstocks were tested under five types of irrigation waters to understand the genetic, physiological, and biochemical bases of salt-tolerance mechanisms. Treatments included control (T1) and four saline water treatments dominant in sodium-sulfate (T2), sodium-chloride (T3), sodium-chloride/sulfate (T4), and calcium/magnesium-chloride/sulfate (T5). T3 caused the highest reduction in survival rate and trunk diameter, followed by T4 and T2, indicating that Na and, to a lesser extent, Cl were the most toxic ions to almond rootstocks. Peach hybrid (Empyrean 1) and peach-almond hybrids (Cornerstone, Bright's Hybrid 5, and BB 106) were the most tolerant to salinity. Rootstock's performance under salinity correlated highly with its leaf Na and Cl concentrations, indicating that Na+ and Cl- exclusion is crucial for salinity tolerance in Prunus. Photosynthetic rate correlated with trunk diameter and proline leaf ratio (T3/T1) significantly correlated with the exclusion of Na+ and Cl-, which directly affected the survival rate. Expression analyses of 23 genes involved in salinity stress revealed that the expression differences among genotypes were closely associated with their performance under salinity. Our genetic, molecular, and biochemical analyses allowed us to characterize rootstocks based on component traits of the salt-tolerance mechanisms, which may facilitate the development of highly salt-tolerant rootstocks.

Expression of the high-affinity K+ transporter 1 (PpHKT1) gene from almond rootstock 'Nemaguard' improved salt tolerance of transgenic Arabidopsis.

Soil salinity affects plant growth and development, which directly impact yield. Plants deploy many mechanisms to cope with, or mitigate, salt stress. One of such mechanism is to control movement of ions from root to shoot by regulating the loading of Na+ in the transpiration stream. The high-affinity K+ transporter 1 (HKT1) is known to play a role in the removal of Na+ from the xylem and bring it back to the root. As almond is a salt-sensitive crop, the rootstock plays an important role in successful almond cultivation in salt-affected regions. We currently lack knowledge on the molecular mechanisms involved in salt tolerance of almond rootstocks. In this study, we complemented the Arabidopsis athkt1 knockout mutant with HKT1 ortholog (PpHKT1) from the almond rootstock 'Nemaguard'. Arabidopsis transgenic lines that were generated in athkt1 background with the constitutive promoter (PpHKT1OE2.2) and the native promoter (PpHKT1NP6) were subjected to different salt treatments. Both transgenic lines survived salt concentrations up to 120 mM NaCl, however, the mutant athkt1 died after 18 days under 120 mM NaCl. At 90 mM NaCl, the dry weight of athkt1 decreased significantly compared to the transgenic lines. Both transgenic lines showed significantly longer lateral roots compared to the athkt1 mutant at 80 mM NaCl treatment. The transgenic lines, PpHKT1OE2.2 and PpHKTNP6 had lower electrolyte leakage and higher relative water content compared to athkt1, suggesting that transgenic plants coped well with increased salt concentration by maintaining the integrity of the membranes. The expression analyses showed that PpHKT1 was induced in PpHKT1OE2.2 and PpHKTNP6 lines under salt treatment, which confirmed that both over-expression and native expression of PpHKT1 in the Arabidopsis mutant can complement salt tolerance function.

Defining and Exploiting Hypersensitivity Hotspots to Facilitate Abscisic Acid Agonist Optimization.

Pyrabactin resistance 1 (PYR1) and related abscisic acid (ABA) receptors are new targets for manipulating plant drought tolerance. Here, we identify and use PYR1 hypersensitive mutants to define ligand binding hotspots and show that these can guide improvements in agonist potency. One hotspot residue defined, A160, is part of a pocket that is occupied by ABA's C6 methyl or by the toluyl methyl of the synthetic agonist quinabactin (QB). A series of QB analogues substituted at the toluyl position were synthesized and provide up to 10-fold gain in activity in vitro. Furthermore, we demonstrate that hypersensitive receptors can be used to improve the sensitivity of a previously described mammalian cell ABA-regulated transcriptional circuit by three orders of magnitude. Collectively, our data show that the systematic mapping of hypersensitivity sites in a ligand-binding pocket can help guide ligand optimization and tune the sensitivity of engineered receptors.

Dynamic control of plant water use using designed ABA receptor agonists.

Drought causes crop losses worldwide, and its impact is expected to increase as the world warms. This has motivated the development of small-molecule tools for mitigating the effects of drought on agriculture. We show here that current leads are limited by poor bioactivity in wheat, a widely grown staple crop, and in tomato. To address this limitation, we combined virtual screening, x-ray crystallography, and structure-guided design to develop opabactin (OP), an abscisic acid (ABA) mimic with up to an approximately sevenfold increase in receptor affinity relative to ABA and up to 10-fold greater activity in vivo. Studies in Arabidopsis thaliana reveal a role of the type III receptor PYRABACTIN RESISTANCE-LIKE 2 for the antitranspirant efficacy of OP. Thus, virtual screening and structure-guided optimization yielded newly discovered agonists for manipulating crop abiotic stress tolerance and water use.

A Rationally Designed Agonist Defines Subfamily IIIA Abscisic Acid Receptors As Critical Targets for Manipulating Transpiration.

Increasing drought and diminishing freshwater supplies have stimulated interest in developing small molecules that can be used to control transpiration. Receptors for the plant hormone abscisic acid (ABA) have emerged as key targets for this application, because ABA controls the apertures of stomata, which in turn regulate transpiration. Here, we describe the rational design of cyanabactin, an ABA receptor agonist that preferentially activates Pyrabactin Resistance 1 (PYR1) with low nanomolar potency. A 1.63 Å X-ray crystallographic structure of cyanabactin in complex with PYR1 illustrates that cyanabactin's arylnitrile mimics ABA's cyclohexenone oxygen and engages the tryptophan lock, a key component required to stabilize activated receptors. Further, its sulfonamide and 4-methylbenzyl substructures mimic ABA's carboxylate and C6 methyl groups, respectively. Isothermal titration calorimetry measurements show that cyanabactin's compact structure provides ready access to high ligand efficiency on a relatively simple scaffold. Cyanabactin treatments reduce Arabidopsis whole-plant stomatal conductance and activate multiple ABA responses, demonstrating that its in vitro potency translates to ABA-like activity in vivo. Genetic analyses show that the effects of cyanabactin, and the previously identified agonist quinabactin, can be abolished by the genetic removal of PYR1 and PYL1, which form subclade A within the dimeric subfamily III receptors. Thus, cyanabactin is a potent and selective agonist with a wide spectrum of ABA-like activities that defines subfamily IIIA receptors as key target sites for manipulating transpiration.

Mechanism of drug resistance in clonally related clinical isolates of Vibrio fluvialis isolated in Kolkata, India.

The molecular mechanisms of drug resistance in 19 strains of Vibrio fluvialis isolated from 1998 to 2002 in Kolkata, India, were investigated. Class 1 integrons were detected in eight strains, and four strains were found to carry SXT integrases. In the presence of carbonyl cyanide m-chlorophenylhydrazone or reserpine, all nalidixic acid- and ciprofloxacin-resistant strains became sensitive, suggesting that drug efflux plays a major role in quinolone resistance in V. fluvialis. It was further seen that strains which had MICs of > 25 microg/ml for nalidixic acid had a sense mutation (Ser to Ile) at position 83 of the quinolone resistance-determining region of gyrA. All except one of the integron- and SXT integrase-bearing strains belonged to the same ribotype.