Regulation of Stomatal Defense by Air Relative Humidity.

It has long been observed that environmental conditions play crucial roles in modulating immunity and disease in plants and animals. For instance, many bacterial plant disease outbreaks occur after periods of high humidity and rain. A critical step in bacterial infection is entry into the plant interior through wounds and natural openings, such as stomata, which are adjustable microscopic pores in the epidermal tissue. Several studies have shown that stomatal closure is an integral part of the plant immune response to reduce pathogen invasion. In this study, we found that high humidity can effectively compromise Pseudomonas syringae-triggered stomatal closure in both Phaseolus vulgaris and Arabidopsis (Arabidopsis thaliana), which is accompanied by early up-regulation of the jasmonic acid (JA) pathway and simultaneous down-regulation of salicylic acid (SA) pathway in guard cells. Furthermore, SA-dependent response, but not JA-dependent response, is faster in guard cells than in whole leaves, suggesting that the SA signaling in guard cells may be independent from other cell types. Thus, we conclude that high humidity, a well-known disease-promoting environmental condition, acts in part by suppressing stomatal defense and is linked to hormone signaling in guard cells.

Drought and salt stress mitigation in crop plants using stress-tolerant auxin-producing endophytic bacteria: a futuristic approach towards sustainable agriculture.

Abiotic stresses, especially drought stress and salt stress in crop plants are accelerating due to climate change. The combined impact of drought and salt is anticipated to lead to the loss of up to 50% of arable land globally, resulting in diminished growth and substantial yield losses threatening food security. Addressing the challenges, agriculture through sustainable practices emerges as a potential solution to achieve Zero Hunger, one of the sustainable development goals set by the IUCN. Plants deploy a myriad of mechanisms to effectively address drought and salt stress with phytohormones playing pivotal roles as crucial signaling molecules for stress tolerance. The phytohormone auxin, particularly indole acetic acid (IAA) emerges as a paramount regulator integral to numerous aspects of plant growth and development. During both drought and salt stress conditions, auxin plays crucial roles for tolerance, but stress-induced processes lead to decreased levels of endogenous free auxin in the plant, leading to an urgent need for auxin production. With an aim to augment this auxin deficiency, several researchers have extensively investigated auxin production, particularly IAA by plant-associated microorganisms, including endophytic bacteria. These endophytic bacteria have been introduced into various crop plants subjected to drought or salt stress and potential isolates promoting plant growth have been identified. However, post-identification, essential studies on translational research to advance these potential isolates from the laboratory to the field are lacking. This review aims to offer an overview of stress tolerant auxin-producing endophytic bacterial isolates while identifying research gaps that need to be fulfilled to utilize this knowledge for the formulation of crop-specific and stress-specific endophyte bioinoculants for the plant to cope with auxin imbalance occurring during these stress conditions.

Purification, characterization of laccase from Pleurotus ostreatus HK35, and optimization for congo red biodecolorization using Box-Behnken design.

This study is the first report on purification, characterization, and application of laccase derived from the white-rot fungus, Pleurotus ostreatus HK35 (Hungary strain), in Congo Red decolorization. The purification process involved ammonium sulfate precipitation, dialysis, anion exchange chromatography, and ultrafiltration, yielding a specific laccase activity of 15.26 U/mg and a 30.21% recovery rate. The purified enzyme, with a molecular weight of approximately 34 kilodaltons, displayed optimal activity at a temperature of 60 °C and pH 4.0 when using 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as a substrate. The enzyme maintained over 82.02 ± 1.01% of its activity at temperatures up to 50 °C after 180 min but displayed less than 5% of its activity at 60 and 70 °C. Notably, the enzyme's activity was significantly enhanced by Pb(NO3)2, whereas ß-mercaptoethanol completely inhibited the activity. Utilizing the Box-Behnken design, we optimized Congo Red decolorization efficiency to 91.05 ± 0.82% at 100 mg/L Congo Red, 1.5 mM mediator concentration, and 1.6 U/mL laccase activity. Analysis of Variance (ANOVA) suggested the model was significant, and all variables significantly influenced decolorization efficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03926-7.

Guard cell purification and RNA isolation suitable for high-throughput transcriptional analysis of cell-type responses to biotic stresses.

Stomata, micro-pores on the leaf surface, are formed by a pair of guard cells. In addition to controlling water loss and gas exchange between the plant and the environment, these cells act as immunity gates to prevent pathogen invasion of the plant apoplast. Here, we report a brief procedure to obtain highly pure guard cell preparations using conditions that preserve the guard cell transcriptome as much as possible for a robust high-throughput RNA sequence analysis. The advantages of this procedure included i) substantial shortening of the time required for obtaining high yield of >97% pure guard cell protoplasts (GCP), ii) extraction of enough high quality RNA for direct sequencing, and iii) limited RNA decay during sample manipulation. Gene expression analysis by reverse transcription quantitative polymerase chain reaction revealed that wound-related genes were not induced during release of guard cells from leaves. To validate our approach, we performed a high-throughput deep-sequencing of guard cell transcriptome (RNA-seq). A total of 18,994 nuclear-encoded transcripts were detected, which expanded the transcriptome by 70%. The optimized GCP isolation and RNA extraction protocols are simple, reproducible, and fast, allowing the discovery of genes and regulatory networks inherent to the guard cells under various stresses.

Escherichia coli O157:H7 induces stronger plant immunity than Salmonella enterica Typhimurium SL1344.

Consumption of fresh produce contaminated with bacterial human pathogens has resulted in various, sometimes deadly, disease outbreaks. In this study, we assessed plant defense responses induced by the fully pathogenic bacteria Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in both Arabidopsis thaliana and lettuce (Lactuca sativa). Unlike SL1344, O157:H7 induced strong plant immunity at both pre-invasion and post-invasion steps of infection. For instance, O157:H7 triggered stomatal closure even under high relative humidity, an environmental condition that generally weakens plant defenses against bacteria in the field and laboratory conditions. SL1344 instead induced a transient stomatal immunity. We also observed that PR1 gene expression was significantly higher in Arabidopsis leaves infected with O157:H7 compared with SL1344. These results suggest that plants may recognize and respond to some human pathogens more effectively than others. Furthermore, stomatal immunity can diminish the penetration of human pathogens through the leaf epidermis, resulting in low bacterial titers in the plant apoplast and suggesting that additional control measures can be employed to prevent food contamination. The understanding of how plant responses can diminish bacterial contamination is paramount in preventing outbreaks and improving the safety of food supplies.

Loss of nucleosome assembly protein 1 affects growth and appressorium structure in blast fungus Magnaporthe oryzae.

Evolutionarily conserved nucleosome assembly protein Nap1 is involved in multiple cellular processes in eukaryotes. In this study, we wanted to explore the role of Nap1 in the life cycle of rice blast fungus Magnaporthe oryzae. The null mutant of M. oryzae NAP1 is viable. However, deletion of NAP1 leads to defects in growth, appressorium morphology, and appressorium turgidity. In the future, plant infection studies can be undertaken to find if these defects lead to compromised virulence of this economically important fungal pathogen.

Early Diverging Fungus Mucor circinelloides Lacks Centromeric Histone CENP-A and Displays a Mosaic of Point and Regional Centromeres.

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high-fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins, were found to co-localize to a short region, one in each of nine large scaffolds, composed of an ∼200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

Stomate-based defense and environmental cues.

Environmental conditions play crucial roles in modulating immunity and disease in plants. For instance, many bacterial disease outbreaks occur after periods of high humidity and rain. A critical step in bacterial infection is entry into the plant interior through wounds or natural openings, such as stomata. Bacterium-triggered stomatal closure is an integral part of the plant immune response to reduce pathogen invasion. Recently, we found that high humidity compromises stomatal defense, which is accompanied by regulation of the salicylic acid and jasmonic acid pathways in guard cells. Periods of darkness, when most stomata are closed, are effective in decreasing pathogen penetration into leaves. However, coronatine produced by Pseudomonas syringae pv. tomato (Pst) DC3000 cells can open dark-closed stomata facilitating infection. Thus, a well-known disease-promoting environmental condition (high humidity) acts in part by suppressing stomatal defense, whereas an anti-stomatal defense factor such as coronatine, may provide epidemiological advantages to ensure bacterial infection when environmental conditions (darkness and insufficient humidity) favor stomatal defense.

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast.

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

An HPLC-based Method to Quantify Coronatine Production by Bacteria.

Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Coronatine Facilitates Pseudomonas syringae Infection of Arabidopsis Leaves at Night.

In many land plants, the stomatal pore opens during the day and closes during the night. Thus, periods of darkness could be effective in decreasing pathogen penetration into leaves through stomata, the primary sites for infection by many pathogens. Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine (COR) and opens stomata, raising an intriguing question as to whether this is a virulence strategy to facilitate bacterial infection at night. In fact, we found that (a) biological concentration of COR is effective in opening dark-closed stomata of Arabidopsis thaliana leaves, (b) the COR defective mutant Pst DC3118 is less effective in infecting Arabidopsis in the dark than under light and this difference in infection is reduced with the wild type bacterium Pst DC3000, and (c) cma, a COR biosynthesis gene, is induced only when the bacterium is in contact with the leaf surface independent of the light conditions. These findings suggest that Pst DC3000 activates virulence factors at the pre-invasive phase of its life cycle to infect plants even when environmental conditions (such as darkness) favor stomatal immunity. This functional attribute of COR may provide epidemiological advantages for COR-producing bacteria on the leaf surface.

Plant innate immunity against human bacterial pathogens.

Certain human bacterial pathogens such as the enterohemorrhagic Escherichia coli and Salmonella enterica are not proven to be plant pathogens yet. Nonetheless, under certain conditions they can survive on, penetrate into, and colonize internal plant tissues causing serious food borne disease outbreaks. In this review, we highlight current understanding on the molecular mechanisms of plant responses against human bacterial pathogens and discuss salient common and contrasting themes of plant interactions with phytopathogens or human pathogens.