Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

The rhizobacterial strain, Pseudomonas putida AKMP7, causes conditional pathogenesis in Arabidopsis thaliana via negative regulation of salicylic acid signaling, under water stress.

We have previously reported a phenomenon of "conditional pathogenesis", in which, a drought-tolerant rhizobacterium, Pseudomonas putida AKMP7, promotes plant growth under well-watered conditions, while, deteriorating plant health under water-stressed conditions, in Arabidopsis thaliana seedlings. To understand the molecular mechanisms behind this phenomenon, we studied the modulation of salicylic acid (SA) biosynthesis as well as SA-responsive gene expression, involved in systemic acquired resistance (SAR), in A. thaliana, by AKMP7, under well-watered and water-stressed conditions. We found that, the plant SA levels were upregulated by AKMP7, both under, well-watered as well as water-stressed conditions. However, the SA signaling gene, Non-expressor of Pathogenesis Related gene 1 (NPR1) and Pathogenesis Related gene 1 (PR1) were upregulated under well-watered conditions and suppressed under water-stress, in AKMP7 inoculated seedlings. To understand the reason for this, we studied the expression of NPR4, a negative regulator of NPR1, and, NPR3, a negative regulator of PR1. We observed that, AKMP7 suppresses NPR1 and, consequently, PR1 genes, by upregulating NPR4 under water stress. To understand the potential role of NPR4 in conditional-pathogenesis, we performed physiological studies with NPR4 knockout mutants of A. thaliana and found that the NPR4 mutants did not exhibit any signs of the characteristic growth retardation caused by AKMP7 inoculation, under water stress. Preliminary studies with the model pathogen, Pseudomonas syringae, indicate that AKMP7 may lead to enhanced disease suppression under well-watered conditions, but not under water-stress. Taken together, our data suggest that, AKMP7 causes conditional pathogenesis by an overall compromise in plant immune response under water-stress.

Native bio-control agents from the rice fields of Telangana, India: characterization and unveiling the potential against stem rot and false smut diseases of rice.

The stem rot caused by Sclerotium hydrophilum and false smut caused by Ustilaginoidea virens are two of the major production constraints in rice cultivation in India and other countries. Stem rot and false smut can be effectively controlled with synthetic fungicides. However, the indiscriminate use of chemical fungicides may cause development of resistance among the pathogens. In addition to this, synthetic fungicides also exhibit harmful impacts on the environment. Exploiting microbe-based alternatives for managing plant diseases diminishes public concerns about the ill effects of pesticide usage in crops. In this regard, the present study was designed to investigate the potential of native microbial biocontrol agents (BCAs) from rice rhizosphere for the sustainable management of stem rot and false smut diseases in rice. Potential BCAs and pathogens were identified and characterized through morphological, biochemical, and sanger sequencing techniques. Bio-efficacy tests of potential BCAs against stem rot and false smut diseases on rice under glasshouse conditions indicated higher seed vigour index of the treated seeds, significant improvement in the growth of the seedling, increased dry weight, reduction in percentage disease index viz., 70.03% (stem rot) and 69.24% (false smut) over the control plants. Phytohormones indole acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA), salicylic acid (SA), and zeatin (tZ) were detected and quantified in the four potential BCAs using liquid chromatography- tandem mass spectrometry (LC-MS/MS). Scanning electron microscopy (SEM) studies revealed the endophytic nature of the strains in rice. The study indicated a positive correlation between the diversity and concentration of phytohormones released by the bioagents and enhanced plant growth promotion and disease suppression in rice.

Probing into the unique relationship between a soil bacterium, Pseudomonas putida AKMP7 and Arabidopsis thaliana: A case of "conditional pathogenesis".

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil bacteria that colonise the rhizosphere and help plants in growth, development, and stress tolerance. While there is a significant body of research elucidating their benefits to plants, studies on the "abnormal" or "unexpected" behavior of these bacteria are almost non-existent. One such study from our laboratory has previously reported a unique situation in which a certain strain of drought and thermo-tolerant PGPR, namely, Pseudomonas putida AKMP7, becomes pathogenic towards Arabidopsis thaliana under drought conditions, but not under normal (well-watered) conditions. In this study, we have probed deeper into this phenomenon of "conditional pathogenesis". We found that, AKMP7 imparts an enhancement in plant growth under well-watered conditions, while, causing a deterioration in plant health under drought conditions. In an attempt to understand the underlying reasons for this phenomenon, we analysed the phytohormones released by Pseudomonas putida AKMP7 using LC-ESI-MS/MS technique. We identified that AKMP7 releases zeatin (a cytokinin), the auxin derivative -indole acetamide and amino acid-conjugates of auxin (indole-3-acetyl-L-alanine, indole-3-acetyl-L-phenylalanine and indole-3-acetyl-L-aspartate) in the growth medium. By treating the plants with commercially obtained forms of these phytohormones, individually or in combination with AKMP7, we identified that zeatin and auxin derivative indole acetamide can play a crucial role in the conditional pathogenesis exhibited by this bacterium on A. thaliana under drought conditions. Our work lays a foundation for further understanding the precise molecular mechanisms involved in this unique phenomenon of conditional/opportunistic pathogenesis.

Genetic, Epigenetic, Genomic and Microbial Approaches to Enhance Salt Tolerance of Plants: A Comprehensive Review.

Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.

A comparative analysis of exopolysaccharide and phytohormone secretions by four drought-tolerant rhizobacterial strains and their impact on osmotic-stress mitigation in Arabidopsis thaliana.

The ability of plant growth promoting rhizobacteria (PGPR) for imparting abiotic stress tolerance to plants has been widely explored in recent years; however, the diversity and potential of these microbes have not been maximally exploited. In this study, we characterized four bacterial strains, namely, Pseudomonas aeruginosa PM389, Pseudomonas aeruginosa ZNP1, Bacillus endophyticus J13 and Bacillus tequilensis J12, for potential plant growth promoting (PGP) traits under osmotic-stress, induced by 25% polyethylene glycol (PEG) in the growth medium. Growth curve analysis was performed in LB medium with or without PEG, in order to understand the growth patterns of these bacteria under osmotic-stress. All strains were able to grow and proliferate under osmotic-stress, although their growth rate was slower than that under non-stressed conditions (LB without PEG). Bacterial secretions were analyzed for the presence of exopolysaccharides and phytohormones and it was observed that all four strains released these compounds into the media, both, under stressed and non-stressed conditions. In the Pseudomonas strains, osmotic stress caused a decrease in the levels of auxin (IAA) and cytokinin (tZ), but an increase in the levels of gibberellic acid. The Bacillus strains on the other hand showed a stress-induced increase in the levels of all three phytohormones. P. aeruginosa ZNP1 and B. endophyticus J13 exhibited increased EPS production under osmotic-stress. While osmotic stress caused a decrease in the levels of EPS in P. aeruginosa PM389, B. tequilensis J12 showed no change in EPS quantities released into the media under osmotic stress when compared to non-stressed conditions. Upon inoculating Arabidopsis thaliana seedlings with these strains individually, it was observed that all four strains were able to ameliorate the adverse effects of osmotic-stress (induced by 25% PEG in MS-Agar medium) in the plants, as evidenced by their enhanced fresh weight, dry weight and plant water content, as opposed to osmotic-stressed, non-inoculated plants.

Modulation of polyamine biosynthesis in Arabidopsis thaliana by a drought mitigating Pseudomonas putida strain.

Plant growth promoting rhizobacteria (PGPR) are a diverse group of beneficial soil bacteria that help plants in myriad ways. They are implicated in the processes of general growth and development, as well as stress mitigation. Although the physiology of plant-PGPR interaction for abiotic stress tolerance has been well reported, the underlying molecular mechanisms in this phenomenon are not clearly understood. Among the many endogenous molecules that have been reported to impart abiotic stress tolerance in plants are a group of aliphatic amines called polyamines. Here, we report the impact of a free living, drought-mitigating rhizobacterial strain, Pseudomonas putida GAP-P45 on the expression of key genes in the polyamine metabolic pathway and the accumulation of the three major polyamines, putrescine, spermidine and spermine in water-stressed Arabidopsis thaliana. We observed that, inoculation of A. thaliana with P. putida GAP-P45 with or without water-stress, caused significant fluctuations in the expression of most polyamine biosynthetic genes (ADC, AIH, CPA, SPDS, SPMS and SAMDC) and cellular polyamine levels at different days of analysis post treatments. The enhanced accumulation of free cellular putrescine and spermidine observed in this study correlated positively with the water stress tolerant phenotype of A. thaliana in response to P. putida GAP-P45 inoculation reported in our previous study (Ghosh et al., 2017). Our data point towards (a) transcriptional regulation of polyamine biosynthetic genes and (b) complex post transcriptional regulation and/or interconversion/canalization of polyamines, by P. putida GAP-P45 under normal and water-stressed conditions.

Impact of heavy metal lead stress on polyamine levels in Halomonas BVR 1 isolated from an industry effluent.

In living systems, environmental stress due to biotic and abiotic factors triggers the production of myriad metabolites as a potential mechanism for combating stress. Among these metabolites are the small polycationic aliphatic amine molecules - polyamines, which are ubiquitous in all living organisms. In this work, we demonstrate a correlation between cellular concentration of three major polyamines (putrescine, spermidine and spermine) with lead exposure on bacteria for a period of 6-24 h. We report that indigenously isolated Halomonas sp. strain BVR 1 exhibits lead induced fluctuations in their cellular polyamine concentration. This response to lead occurs within 6 h post metal treatment. During the same time interval there was a surge in the growth of bacteria along with an enhancement in the putrescine levels. We conclude that in Halomonas sp. strain BVR 1, an early response is seen with respect to modulation of polyamines as a result of lead treatment and hypothesize that endogenous polyamines contribute towards scavenging lead in these bacteria.

Biosynthetic approach for functional protein microarrays.

Protein microarrays have emerged as an indispensable research tool for providing information about protein functions and interactions through high-throughput screening. Traditional methods for immobilizing biomolecules onto solid surfaces have been based on covalent and noncovalent binding, entrapment in semipermeable membranes, microencapsulation, sol gel, and hydrogel methods. Each of these techniques has its own strengths but fails to combine the most important tenets of a functional protein microarray such as covalent attachment, native protein conformation, homogeneity of the protein monolayer, control over active site orientation, and retention of protein activity. Here we present a selective and site-directed covalent immobilization technique for proteins via a benzoxazine ring formation through a Diels-Alder reaction in water and a genetically encoded 3-amino-L-tyrosine (3-NH(2)Tyr) amino acid. Fully functional protein microarrays, with monolayer arrangements and complete control over their orientations, were generated using this strategy.

The response of high and low polyamine-producing cell lines to aluminum and calcium stress.

The diamine putrescine (Put) has been shown to accumulate in tree leaves in response to high Al and low Ca in the soil, leading to the suggestion that this response may provide a physiological advantage to leaf cells under conditions of Al stress. The increase in Put is reversed by Ca supplementation in the soil. Using two cell lines of poplar (Populus nigra x maximowiczii), one with constitutively high Put (resulting from transgenic expression of a mouse ornithine decarboxylase--called HP cells) and the other with low Put (control cells), we investigated the effects of reduced Ca (0.2-0.8 mM vs. 4 mM) and treatment with 0.1 mM Al on several biochemical parameters of cells. We found that in the presence of reduced Ca concentration, the HP cells were at a disadvantage as compared to control cells in that they showed greater reduction in mitochondrial activity and a reduction in the yield of cell mass. Upon addition of Al to the medium, the HP cells, however, showed a reversal of low-Ca effects. We conclude that due to increased ROS production in the HP cells, their tolerance to low Ca is compromised. Contrary to the expectation of deleterious effects, the HP cells showed an apparent advantage in the presence of Al in the medium, which could have come from reduced uptake of Al, enhanced extrusion of Al following its accumulation, and perhaps a reduction in Put catabolism as a result of a reduction in its biosynthesis.

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids.

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.

Putrescine overproduction negatively impacts the oxidative state of poplar cells in culture.

While polyamines (PAs) have been suggested to protect cells against Reactive Oxygen Species (ROS), their catabolism is known to generate ROS. We compared the activities of several enzymes and cellular metabolites involved in the ROS scavenging pathways in two isogenic cell lines of poplar (Populus nigraxmaximowiczii) differing in their PA contents. Whereas the control cell line was transformed with beta-glucuronidase (GUS), the other, called HP (High Putrescine), was transformed with a mouse ornithine decarboxylase (mODC) gene. The expression of mODC resulted in several-fold increased production of putrescine as well its enhanced catabolism. The two cell lines followed a similar trend of growth over the seven-day culture cycle, but the HP cells had elevated levels of soluble proteins. Accumulation of H(2)O(2) was higher in the HP cells than the control cells, and so were the activities of glutathione reductase and monodehydroascorbate reductase; the activity of ascorbate peroxidase was lower in the former. The contents of reduced glutathione and glutamate were significantly lower in the HP cells but proline was higher on some days of analysis. There was a small difference in mitochondrial activity between the two cell lines, and the HP cells showed increased membrane damage. In the HP cells, increased accumulation of Ca was concomitant with lower accumulation of K. We conclude that, while increased putrescine accumulation may have a protective role against ROS in plants, enhanced turnover of putrescine actually can make them vulnerable to increased oxidative damage.