Plant Colonization Research Articles

Transcriptomic profiling of 'Candidatus Liberibacter asiaticus' in different citrus tissues reveals novel insights into Huanglongbing pathogenesis.

'Candidatus Liberibacter asiaticus' (Las) is a gram-negative bacterial pathogen associated with citrus huanglongbing (HLB) or greening disease. Las is transmitted by the Asian citrus psyllid (ACP) where it colonizes the phloem tissue, resulting in substantial economic losses to citrus industry worldwide. Despite extensive efforts, effective management strategies against HLB remain elusive, necessitating a deeper understanding of the pathogen's biology. Las undergoes cell-to-cell movement through phloem flow and colonizes different tissues in which Las may have varying interactions with the host. Here, we investigate the transcriptomic landscape of Las in citrus seed coat vasculatures, enabling a complete gene expression profiling of Las genome and revealing unique transcriptomic patterns compared to previous studies using midrib tissues. Comparative transcriptomics between seed coat, midrib and ACP identified specific responses and metabolic states of Las in different host tissue. Two Las virulence factors that exhibit higher expression in seed coat can suppress callose deposition. Therefore, they may contribute to unclogging sieve plate pores during Las colonization in seed coat vasculature. Furthermore, analysis of regulatory elements uncovers a potential role of LuxR-type transcription factors in regulating Liberibacter effector gene expression during plant colonization. Together, this work provides novel insights into the pathogenesis of the devastating citrus HLB.

Pseudomonas rhodesiae HAI-0804 suppresses Pythium damping off and root rot in cucumber by its efficient root colonization promoted by amendment with glutamate

Plant diseases caused by soil-borne fungi and oomycetes significantly reduce yield and quality of many crops in the agricultural systems and are difficult to control. We herein examine Pseudomonas rhodesiae HAI-0804, a bacterial biological control agent that was originally developed for control of bacterial diseases on the surface of vegetables, and assessed its efficacy at controlling soil-borne diseases caused by oomycetes. Strain HAI-0804 did not exhibit detectable antibiotic activity toward Pythium ultimum, a causal agent of damping-off and root rot; however, it effectively protected against Pythium damping-off and root rot in cucumber. Exogenous glutamate enhanced the efficacy of biocontrol, the production of siderophore pyoverdine, root colonization in cucumber plants, and the ratio of biofilm formation to planktonic cells. The epiphytic fitness of strain HAI-0804 appears to contribute to plant protection efficacy against a broad spectrum of pathogens for both above-ground plant parts and the rhizosphere.

Combined Effect of Arbuscular Mycorrhizal Fungi and Bradyrhizobium japonicum on Growth, Yield and Nodulation in Soybean

Response of soybean for combined application of AM fungi and Bradyrhizobium japonicum were studied in pot culture experiment in completely randomized design (CRD). Dual inoculation of AM fungi and B. japonicum improved growth, yield, AMF colonization and nodulation in soybean plant, in comparison to single inoculation. Increase in the level of AMF inoculation promoted the growth significantly. Dry weight of the plant was 5.54 g/ plant and 6.02 g/ plant in plants treated with B. japonicum and AMF respectively, which significantly increased to 7.36 g/ plant up on combined application. Similar results were obtained in plant height, No. of pods /plant, seed index, nodulation, AMF density and colonization and chlamydospore population in soybean rhizosphere. Thus, the experiment signifies the importance of combined application for the improvement of crop growth and yield.

Within-Site Variations in Soil Physicochemical Properties Explained the Spatiality and Cohabitation of Arbuscular Mycorrhizal Fungi in the Roots of Cryptomeria Japonica

Arbuscular mycorrhizal fungi (AMF) live in a community in the roots of host plants. Still, the patterns and factors that drive their spatiality and cohabitation remain uncovered, particularly that of trees in planted forests, which we aimed to clarify in Cryptomeria japonica, a major plantation tree in Japan. We analyzed 65 paired root and soil samples of Cryptomeria japonica trees collected from 11 microsite (MS) plots at two environmentally different forest sites in central Japan and measured soil pH, total phosphorus (TP), C, N, and the carbon-to-nitrogen ratio. Root AMF communities were recovered using Illumina’s next-generation amplicon sequencing targeting the small subunit of ribosomal DNA. We detected more than 500 AMF OTUs at each site but only three belonging to Dominikia, Rhizophagus, and Sclerocystis were dominant in the roots of C. japonica, detected each at an average relative abundance higher than 20%. Two showed negatively correlated spatial distributions and different associations with soil pH. Similarly, the physicochemical properties at MSs significantly determined the AMF assemblages in the roots of C. japonica. Dominikia, Rhizophagus, and Sclerocystis coexist in the roots of C. japonica where soil physicochemical properties, particularly pH, determine their spatial dynamic, turnovers, and cohabitation patterns. These findings highlight the importance of simultaneous colonization of plants by multiple AMF.

Copper acquisition is essential for plant colonization and virulence in a root-infecting vascular wilt fungus.

Plant pathogenic fungi provoke devastating agricultural losses and are difficult to control. How these organisms acquire micronutrients during growth in the host environment remains poorly understood. Here we show that efficient regulation of copper acquisition mechanisms is crucial for plant colonization and virulence in the soilborne ascomycete Fusarium oxysporum, the causal agent of vascular wilt disease in more than 150 different crops. Using a combination of RNA-seq and ChIP-seq, we establish a direct role of the transcriptional regulator Mac1 in activation of copper deficiency response genes, many of which are induced during plant infection. Loss of Mac1 impaired growth of F. oxysporum under low copper conditions and abolishes pathogenicity on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, overexpression of two Mac1 target genes encoding a copper reductase and a copper transporter was sufficient to restore virulence in the mac1 mutant background. Our results establish a previously unrecognized role of copper reduction and uptake in fungal infection of plants and reveal new ways to protect crops from phytopathogens.

Effect of Drought Stress During Seed Development on Charcoal Rot and Yield of Soybean.

The primary controls for charcoal rot in soybean, caused by the fungal pathogen Macrophomina phaseolina, are to avoid drought stress and to plant a moderately resistant cultivar. The effects of irrigation and cultivar were determined in 2011 and 2013 at the Lon Mann Cotton Research Station, Marianna, AR. Four soybean cultivars (Hutcheson, Osage, Ozark, and R01581F) were planted in plots with or without added M. phaseolina inoculum and subjected to three furrow irrigation regimes: full-season irrigation (Full), irrigation terminated at R5 (CutR5), and nonirrigated (NonIrr). Normalized difference vegetation index (NDVI) was measured at R3 and R6. At harvest, plants and yields were collected. Roots and stems were split and the extent of visible colonization by M. phaseolina microsclerotia was assessed in the roots with a 1 to 5 scale (RSS) and the percent plant height stem discoloration measured. Precipitation in September and October was 54 and 65% below the 30-year average in 2011 and 2013, respectively. The CutR5 irrigation treatment resulted in one less irrigation than Full each year, but CutR5 NDVIs at R6 and yields were significantly lower than those with Full and not significantly different from those of NonIrr. The CutR5 RSS ratings were greater than either Full or NonIrr. Plant colonization by M. phaseolina was negatively correlated to yield in 2011 but not in 2013. No premature plant death caused by charcoal rot was observed in either year. These results indicated that late-season drought stress may be more important to charcoal rot development than drought stress throughout the season, but other factors are needed to trigger early plant death and subsequent yield losses observed in grower fields.

Changing dominance of invasive Phragmites australis and native plant colonization with variation in management, wildfires, and soils in a desert wetland

Abstract Among the most widely distributed species globally, common reed (Phragmites australis (Cav.) Trin. ex Steud.) has generated extensive interest in invasive plant science and management because its introduced strains are highly invasive and often form monocultures that alter ecosystem properties. In desert wetlands in Las Vegas, Nevada, USA, where management goals included reducing hazardous P. australis fuels and increasing native plant diversity, we assessed variation in P. australis cover, the degree of native plant colonization, and soil seed banks after P. australis management treatments (cutting, glyphosate-imazapyr herbicide) and wildfires across gradients in soil properties. Based on change in P. australis cover during six measurement events over 24 months, 24 study sites formed three groups: 1) decreasing cover, where initially high P. australis cover (60-85%) decreased to < 5% following multiple cutting or herbicide treatments; 2) sustaining low cover, where wildfire or clearing was associated with initially low P. australis cover which remained low (< 30%) after multiple herbicide applications; and 3) sustaining high cover (45-100% initially and remaining 30-100%), including sites unmanaged or treated/burned only once. High soil salinity correlated with low post-management P. australis cover. No native plants were detected in the sustaining high P. australis cover group, despite natives occurring in the seed bank. Where management reduced P. australis cover, minimal native plant colonization did occur. Secondary invasion by other non-native plants was nearly absent. Our results suggest that if P. australis can be initially cleared, multiple herbicide applications can persistently keep cover low, especially on drier, saline soils. Slow native plant colonization suggests that a phased approach may be useful to initially reduce P. australis cover, keep it low via repeated treatments, and actively revegetate sites with native species tailored to the moisture-salinity gradient across P. australis-invaded habitats.

Phosphonates of Pectobacterium atrosepticum: Discovery and Role in Plant-Pathogen Interactions.

Many phytopathogens' gene products that contribute to plant-pathogen interactions remain unexplored. In one of the most harmful phytopathogenic bacterium Pectobacterium atrosepticum (Pba), phosphonate-related genes have been previously shown to be among the most upregulated following host plant colonization. However, phosphonates, compounds characterized by a carbon-phosphorus bond in their composition, have not been described in Pectobacterium species and other phytopathogenic bacteria, with the exception of Pseudomonas syringae and Pantoea ananatis. Our study aimed to determine whether Pba synthesizes extracellular phosphonates and, if so, to analyze their physiological functions. We demonstrated that Pba produces two types of extracellular phosphonates: 2-diethoxyphosphorylethanamine and phenylphosphonic acid. Notably, such structures have not been previously described among natural phosphonates. The production of Pba phosphonates was shown to be positively regulated by quorum sensing and in the presence of pectic compounds. Pba phosphonates were found to have a positive effect on Pba stress resistance and a negative effect on Pba virulence. The discovered Pba phosphonates are discussed as metabolites that enable Pba to control its "harmful properties", thereby maintaining its ecological niche (the host plant) in a relatively functional state for an extended period.

Influence of Enhanced Synthesis of Exopolysaccharides in Rhizobium ruizarguesonis and Overproduction of Plant Receptor to these Compounds on Colonizing Activity of Rhizobia in Legume and Non-Legume Plants and Plant Resistance to Phytopathogenic Fungi.

Rhizobial exopolysaccharides (EPS) may provide stabilization of membranes against external factors, as well as improved surface adhesion, but their role in interaction with legume and non-legume plants is still far from understanding. In this work, the transcriptional regulator RosR of Rhizobium ruizarguesonis, which regulates the synthesis of EPS, was overproduced in a pHC60 plasmid and expressed in the RCAM 1026 strain. This resulted in an improved production of EPS by this recombinant strain. Comparative analysis of the inoculation of pea Pisum sativum plants with R. ruizarguesonis pHC60-rosR and strain carrying the empty plasmid revealed an essential increase in the number of nodules, root length and biomass in plants inoculated with this EPS-overproducing strain. It demonstrates that the enhanced EPS synthesis by rhizobia may stimulate plant root colonization and subsequent nodule formation in pea plants. The influence of enhanced EPS synthesis in rhizobia on colonizing activity was also estimated in non-legume plant tomato Solanum lycopersicum. Our findings shown the increased colonization of the root surface and stimulation of the shoot biomass of inoculated plants. Inoculation of pea and tomato with EPS-overproducing rhizobial strain essentially increased plant resistance to phytopathogenic fungi Fusarium culmorum and F. oxysporum in both legume and non-legume plants, demonstrating a significant biocontrol effect of this recombinant strain. Furthermore, we have identified the PsLYK10 gene that encodes a putative EPS receptor in P. sativum, although no significant effect of PsLYK10 overexpression on nodulation in legume (pea P. sativum) and colonization of roots of non-legume plants by rhizobia was found compared to enhanced production of EPS by rhizobia.

Mitigation of microplastic toxicity in soybean by synthetic bacterial community and arbuscular mycorrhizal fungi interaction: Altering carbohydrate metabolism, hormonal transduction, and genes associated with lipid and protein metabolism

Microplastics (MPs) have become ubiquitous environmental pollutants, eliciting concerns about their negative impacts on terrestrial and agroecosystems. However, employing sustainable remediation approaches holds the potential to mitigate these negative impacts and bolster MPs tolerance in both the seeds and vegetative structures of plants. The current investigation explores the impact of arbuscular mycorrhizal fungi (AMF) and synthetic bacterial community (SynCom) as potential alleviators of MP-induced stress. Results exhibited that co-applied SynCom and AMF, resulted in the highest plant biomass, leaf area, pod per plant, and 100-seed weight under control (no MPs) and MPs stress. The combined application of SynCom and AMF enhanced colonization, association, and arbuscule abundance in the non-stressed plants (no MPs) as compared to stressed plants. The sole or co-application of SynCom and AMF enhanced the primary metabolites content and mitigated the MPs-induced reduction in soluble sugars, lipids, protein, and oil contents. Soybean inoculation stimulated the genes expression involved in lipid and protein biosynthesis, while a contradictory drift was noted for genes associated with lipid and protein degradation, underpinning the observed increment in protein and lipid content. Soybean inoculated with AMF exhibited the utmost α-amylase and β-amylase activities, demonstrating enhanced osmolyte (soluble sugar) production, mainly under MPs stress. Remarkably, sole or co-application further reinforces the positive effect of MPs stress on the osmoprotectants and antioxidant levels, for instance, phenol, flavonoid, glycine betaine contents, and glutathione-S-transferase (GST) activities. Subsequent to the stress release, a stress hormones known as abscisic acid (ABA) reduced in the seeds of inoculated soybean while gibberellin (GA), trans-zeatin riboside (ZR), and indole acetic acid (IAA) were increased. Therefore, it is concluded that the combined application of SynCom and AMF protected the soybean plants from MPs-induced oxidative damage by upregulating osmoprotectants and antioxidant levels. This study provides insights into the potential of sustainable remediation approaches in alleviating the complex impact of MPs on soybean, contributing to future strategies for environmental sustainability. Future studies could explore the optimization of SynCom and AMF formulations for different crops and environmental conditions, potentially leading to practical applications in sustainable agriculture and pollution management.

Endophyte Bacillus vallismortis BL01 to Control Fungal and Bacterial Phytopathogens of Tomato (Solanum lycopersicum L.) Plants

Some strains of Bacillus vallismortis have been reported to be efficient biocontrol agents against tomato pathogens. The aim of our study was to assess the biocontrol ability of the endophytic strain BL01 Bacillus vallismortis through in vitro and field trials, as well as to verify its plant colonization ability and analyze the bacterial genome in order to find genes responsible for the biocontrol activity. We demonstrated in a gnotobiotic system and by confocal laser microscopy that the endophytic strain BL01 was able to colonize the endosphere and rhizosphere of tomato, winter wheat and oilseed rape. In vitro experiments demonstrated the inhibition activity of BL01 against a wide range of phytopathogenic fungi and bacteria. BL01 showed biological efficacy in two-year field experiments with tomato plants against black bacterial spotting by 40–70.8% and against late blight by 47.1% and increased tomato harvest by 24.9% or 10.9 tons per hectare compared to the control. Genome analysis revealed the presence of genes that are responsible for the synthesis of biologically active secondary metabolites, which could be responsible for the biocontrol action. Strain BL01 B. vallismortis can be considered an effective biocontrol agent to control both fungal and bacterial diseases in tomato plants.

Biocontrol potential of Bacillus velezensis SEC-024A against southern blight of industrial hemp

Agroathelia rolfsii is the causative agent of southern blight of industrial hemp, which is a soil-borne infection resulting in grave economic losses in industrial hemp (Cannabis sativa L.). Chemical pesticides are currently the primary means of controlling this disease, but their use is harmful to human health as well as to the environment. Urgent action is needed to screen eco-friendly biocontrol agents against this pathogen. An endophytic bacterium SEC-024A was isolated from the leaf tissue of industrial hemp and exhibited a significant antagonistic effect on A. rolfsii, with a plate inhibition rate of 80.5 % and a pot control effect of 74.1 %. Through whole genome sequencing, SEC-024A was identified to be Bacillus velezensis. It contained multiple gene clusters involved in secondary metabolite biosynthesis and the genes that promote plant growth, colonization, and pathogen defense. Bacillus velezensis SEC-024A produces volatile organic compounds (VOCs) that serve as its primary fungistatic agents. Nine VOCs produced by SEC-024A were detected through gas chromatography-mass spectrometry. These VOCs were individually tested for their antifungal effects. Four volatiles, namely isobutyric acid, tiglic acid, trans-2-octenal, and 2-decanone, exhibited notable inhibitory effects on A. rolfsii with EC50 values of 0.012, 0.038, 0.009, and 0.046 μL/mL, respectively. Additionally, trans-2-octenal demonstrated broad-spectrum antifungal activity against various soil-borne pathogens. Together, this study suggests that B. velezensis SEC-024A holds promise as a novel microbial agent for improving plant growth and controlling diseases.

Evolution of gene networks underlying adaptation to drought stress in the wild tomato Solanum chilense.

Drought stress is a key limitation for plant growth and colonization of arid habitats. We study the evolution of gene expression response to drought stress in a wild tomato, Solanum chilense, naturally occurring in dry habitats in South America. We conduct a transcriptome analysis under standard and drought experimental conditions to identify drought-responsive gene networks and estimate the age of the involved genes. We identify two main regulatory networks corresponding to two typical drought-responsive strategies: cell cycle and fundamental metabolic processes. The metabolic network exhibits a more recent evolutionary origin and a more variable transcriptome response than the cell cycle network (with ancestral origin and higher conservation of the transcriptional response). We also integrate population genomics analyses to reveal positive selection signals acting at the genes of both networks, revealing that genes exhibiting selective sweeps of older age also exhibit greater connectivity in the networks. These findings suggest that adaptive changes first occur at core genes of drought response networks, driving significant network re-wiring, which likely underpins species divergence and further spread into drier habitats. Combining transcriptomics and population genomics approaches, we decipher the timing of gene network evolution for drought stress response in arid habitats.

Distinct contributions of microbial and plant residues to SOC during ecosystem primary succession in a Tibetan glacier foreland

Soil organic carbon (SOC) rapidly accumulates during ecosystem primary succession in glacier foreland. This makes it an ideal model for studying soil carbon sequestration and stabilization, which are urgently needed to mitigate climate change. Here, we investigated SOC dynamics in the Kuoqionggangri glacier foreland on the Tibetan Plateau. The study area along a deglaciation chronosequence of 170-year comprising three ecosystem succession stages, including barren ground, herb steppe, and legume steppe. We quantified amino sugars, lignin phenols, and relative expression of genes associated with carbon degradation to assess the contributions of microbial and plant residues to SOC, and used FT-ICR mass spectroscopy to analyze the composition of dissolved organic matter. We found that herbal plant colonization increased SOC by enhancing ecosystem gross primary productivity, while subsequent legumes development decreased SOC, due to increased ecosystem respiration from labile organic carbon inputs. Plant residues were a greater contributor to SOC than microbial residues in the vegetated soils, but they were susceptible to microbial degradation compared to the more persistent and continuously accumulating microbial residues. Our findings revealed the organic carbon accumulation and stabilization process in early soil development, which provides mechanism insights into carbon sequestration during ecosystem restoration under climate change.

Synergism or Antagonism: Do Arbuscular Mycorrhizal Fungi and Plant Growth-Promoting Rhizobacteria Work Together to Benefit Plants?

In agriculture, abiotic and biotic stress reduce yield by 51–82% and 10–16%, respectively. Applications of biological agents such as plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) can improve plant growth. Applications of lone PGPR and AMF also help plants resist abiotic and biotic stressors. The reports for dual inoculation of AMF and PGPR to benefit plants and tackle stressors are largely unknown. It is speculated that PGPR colonization in plants enhances AMF infection during dual AMF and PGPR application, although increased AMF colonization does not always correlate with the increased benefits for the plant hosts. Further research is needed regarding molecular mechanisms of communication during dual inoculations, and dual-inoculation enhancement of induced systemic resistance under pathogen stress, to understand how dual inoculations can result in enhanced plant benefits. The influence of application timing of AMF and PGPR dual inoculations on mitigating abiotic and biotic stress is also not well understood. This review documents the factors that govern and modulate the dual application of AMF and PGPR for plant benefits against stress responses, specifically abiotic (drought) stress and stress from pathogen infection.

Evolution of phosphate scouting in the terrestrial biosphere.

Chemistry assigns phosphorus and its most oxidized form, inorganic phosphate, unique roles for propelling bioenergetics and metabolism in all domains of life, possibly since its very origin on prebiotic Earth. For plants, access to the vital mineral nutrient profoundly affects growth, development and vigour, thus constraining net primary productivity in natural ecosystems and crop production in modern agriculture. Unlike other major biogenic elements, the low abundance and uneven distribution of phosphate in Earth's crust result from the peculiarities of phosphorus cosmochemistry and geochemistry. Here, we trace the chemical evolution of the element, the geochemical phosphorus cycle and its acceleration during Earth's history until the present (Anthropocene) as well as during the evolution and rise of terrestrial plants. We highlight the chemical and biological processes of phosphate mobilization and acquisition, first evolved in bacteria, refined in fungi and algae and expanded into powerful phosphate-prospecting strategies during land plant colonization. Furthermore, we review the evolution of the genetic and molecular networks from bacteria to terrestrial plants, which monitor intracellular and extracellular phosphate availabilities and coordinate the appropriate responses and adjustments to fluctuating phosphate supply. Lastly, we discuss the modern global phosphorus cycle deranged by human activity and the challenges imposed ahead. This article is part of the theme issue 'Evolution and diversity of plant metabolism'.

A membrane localized RTX-like protein mediates physiochemical properties of the Pantoea stewartii subsp. stewartii cell envelope that impact surface adhesion, cell surface hydrophobicity and plant colonization

Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart’s wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss’ colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.

RIN4 immunity regulators mediate recognition of the core effector RipE1 of Ralstonia solanacearum by the receptor Ptr1.

Ralstonia solanacearum causes lethal bacterial wilt diseases in numerous crops, resulting in considerable yield losses. Harnessing genetic resistance is desirable for safeguarding plants against phytopathogens. However, genetic resources resistant to bacterial wilt are limited in crops. RipE1, a conserved type Ⅲ effector with cysteine protease activity, is recognized in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana). Here, using a virus-induced gene silencing approach, we identified the gene encoding N. benthamiana homologue of Ptr1 (NbPtr1a), a coiled-coil nucleotide-binding leucine-rich repeat receptor (NLR) recognizing RipE1. Silencing or editing NbPtr1a completely abolished RipE1-induced cell death, indicating recognition of RipE1 by NbPtr1a. Genetic complementation confirmed this recognition, which is conserved across multiple solanaceous plants. Expression of RipE1 in planta or within pathogenic bacteria promoted pathogen colonization of Nbptr1a mutant plants, demonstrating its virulence function independent of NLR recognition. Silencing NbRIN4 enhanced RipE1-induced cell death, while expressing NbRIN4 inhibited it, suggesting that NbRIN4 is involved in recognition of NbPtr1a-RipE1. Furthermore, RipE1 associated with and cleaved NbRIN4, AtRIN4, and tomato (Solanum lycopersicum) SlRIN4 proteins through its cysteine protease activity. Silencing NbRIN4 in Nbptr1a mutants did not prevent RipE1 from promoting pathogen colonization, suggesting that NbRIN4 is not the primary target for RipE1-mediated virulence. Additionally, NbRIN4 suppressed self-association of the coiled-coil domain of NbPtr1a, which is critical for NbPtr1a-mediated cell death and resistance. Finally, we demonstrated that activation of NbPtr1a requires RipE1-mediated elimination of NbRIN4. Given the conserved nature of RipE1, Ptr1 holds great potential for protecting crops from diverse R. solanacearum strains and other distinct pathogens.

Assembly and evaluation of a confocal microscopy image analysis pipeline useful in revealing the secrets of plant-fungal interactions

The ability of laser scanning confocal microscopy to generate high-contrast 2D and 3D images has become essential in studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins has several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging pipeline that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This pipeline relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability, allowing the different stains to penetrate and effectively bind to their targets, resulting in a consistent visualization of cellular structures. To expand the utility of this pipeline, we used the staining techniques in conjunction with machine learning to analyze fungal biomass through volume analysis, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This pipeline is simple to use, robust, consistent across host and fungal species and can be applied to most plant-fungal interactions. Therefore, this pipeline can be used to characterize model systems as well as non-model interactions where transformation is not routine.

Effect of Drought and Rehydration on Physiological Characteristics of Agriophyllum squarrosum (L.) Moq. in Different Habitats.

Agriophyllum squarrosum (L.) Moq. is a highly prevalent xerophytic species found throughout northern China. It is suitable for cultivation in semi-arid sandy environments and may establish roots in arid desert locations. This species plays a pioneering and exploratory role in the colonization of desert plants. In this study, we selected A. squarrosum from the Urat desert steppe (UD) and Horqin sandy land (HS) to explore their adaptation mechanisms to drought and rehydration environments by using the pot weighing control method to simulate an arid environment. The findings showed that the control (watering to 60-65% of field capacity) exceeded its required amount and the leaves turned yellow. The chlorophyll content was lower than those under moderate and severe drought, and rehydration caused a decrease. However, the contents of malondialdehyde, soluble sugar, and proline in the drought treatment were higher than those in the control. Under moderate and severe drought, the chlorophyll content and the quantum efficiency of photosystem II (Fv/Fm) of A. squarrosum from UD were higher than those from HS. During drought and rehydration processes, the proline content was relatively lower, while the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) and the content of soluble sugar and soluble protein were higher. However, antioxidant enzymes and osmoregulators from UD were higher than those from HS. The results suggest that the stronger ability of A. squarrosum to endure drought environments in UD is due to the high level of antioxidant enzymes and osmoregulators, which are conducive to relieving cell membrane damage when subjected to drought and rehydration.