Regulation of Bacterial Growth and Behavior by Host Plant.

Plants are associated with diverse bacteria in nature. Some bacteria are pathogens that decrease plant fitness, and others are beneficial bacteria that promote plant growth and stress resistance. Emerging evidence also suggests that plant-associated commensal bacteria collectively contribute to plant health and are essential for plant survival in nature. Bacteria with different characteristics simultaneously colonize plant tissues. Thus, plants need to accommodate bacteria that provide service to the host plants, but they need to defend against pathogens at the same time. How do plants achieve this? In this review, we summarize how plants use physical barriers, control common goods such as water and nutrients, and produce antibacterial molecules to regulate bacterial growth and behavior. Furthermore, we highlight that plants use specialized metabolites that support or inhibit specific bacteria, thereby selectively recruiting plant-associated bacterial communities and regulating their function. We also raise important questions that need to be addressed to improve our understanding of plant-bacteria interactions.

Decomposition of dynamic transcriptomic responses during effector-triggered immunity reveals conserved responses in two distinct plant cell populations.

Rapid plant immune responses in the appropriate cells are needed for effective defense against pathogens. Although transcriptome analysis is often used to describe overall immune responses, collection of transcriptome data with sufficient resolution in both space and time is challenging. We reanalyzed public Arabidopsis time-course transcriptome data obtained after low-dose inoculation with a Pseudomonas syringae strain expressing the effector AvrRpt2, which induces effector-triggered immunity in Arabidopsis. Double-peak time-course patterns are prevalent among thousands of upregulated genes. We implemented a multi-compartment modeling approach to decompose the double-peak pattern into two single-peak patterns for each gene. The decomposed peaks reveal an "echoing" pattern: the peak times of the first and second peaks correlate well across most upregulated genes. We demonstrated that the two peaks likely represent responses of two distinct cell populations that respond either cell autonomously or indirectly to AvrRpt2. Thus, the peak decomposition has extracted spatial information from the time-course data. The echoing pattern also indicates a conserved transcriptome response with different initiation times between the two cell populations despite different elicitor types. A gene set highly overlapping with the conserved gene set is also upregulated with similar kinetics during pattern-triggered immunity. Activation of a WRKY network via different entry-point WRKYs can explain the similar but not identical transcriptome responses elicited by different elicitor types. We discuss potential benefits of the properties of the WRKY activation network as an immune signaling network in light of pressure from rapidly evolving pathogens.

Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

How plants manage pathogen infection.

To combat microbial pathogens, plants have evolved specific immune responses that can be divided into three essential steps: microbial recognition by immune receptors, signal transduction within plant cells, and immune execution directly suppressing pathogens. During the past three decades, many plant immune receptors and signaling components and their mode of action have been revealed, markedly advancing our understanding of the first two steps. Activation of immune signaling results in physical and chemical actions that actually stop pathogen infection. Nevertheless, this third step of plant immunity is under explored. In addition to immune execution by plants, recent evidence suggests that the plant microbiota, which is considered an additional layer of the plant immune system, also plays a critical role in direct pathogen suppression. In this review, we summarize the current understanding of how plant immunity as well as microbiota control pathogen growth and behavior and highlight outstanding questions that need to be answered.

Temporally-coordinated bivalent histone modifications of BCG1 enable fungal invasion and immune evasion.

Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.

Commensal lifestyle regulated by a negative feedback loop between Arabidopsis ROS and the bacterial T2SS.

Despite the plant health-promoting effects of plant microbiota, these assemblages also comprise potentially detrimental microbes. How plant immunity controls its microbiota to promote plant health under these conditions remains largely unknown. We find that commensal bacteria isolated from healthy Arabidopsis plants trigger diverse patterns of reactive oxygen species (ROS) production dependent on the immune receptors and completely on the NADPH oxidase RBOHD that selectively inhibited specific commensals, notably Xanthomonas L148. Through random mutagenesis, we find that L148 gspE, encoding a type II secretion system (T2SS) component, is required for the damaging effects of Xanthomonas L148 on rbohD mutant plants. In planta bacterial transcriptomics reveals that RBOHD suppresses most T2SS gene expression including gspE. L148 colonization protected plants against a bacterial pathogen, when gspE was inhibited by ROS or mutation. Thus, a negative feedback loop between Arabidopsis ROS and the bacterial T2SS tames a potentially detrimental leaf commensal and turns it into a microbe beneficial to the host.

Divide and conquer: Spatiotemporal plant innate immunity at single-cell resolution.

Plants have evolved an innate immune system to cope with devastating plant diseases jeopardizing food security. In this issue of Cell Host and Microbe, Tang et al. use single-cell approaches to disentangle spatiotemporal dynamics and cell-type-specific functionalities of plant immunity, providing strategies for precise crop engineering.

Expression of a mycoparasite protease in plant petals suppresses the petal-mediated infection by necrotrophic pathogens.

Sclerotinia sclerotiorum and Botrytis cinerea are necrotrophic plant-pathogenic fungi, causing substantial economic losses on many crops. So far, resistant cultivars against these pathogens are unavailable in most crops. Here, we show that the serine protease CmSp1 of Coniothyrium minitans, a well-characterized mycoparasite of S. sclerotiorum, contributed to suppressing the petal-mediated infection by S. sclerotiorum in rapeseed. Application of recombinant CmSp1 proteins facilitates the bulk degradation of S. sclerotiorum proteins and inhibits spore germination and hyphal growth of S. sclerotiorum and B. cinerea, thereby preventing the development of both diseases. Stable transgenic rapeseed plants with tissue-specific expression of CmSp1 in flower petals inhibit the petal-mediated infection by both S. sclerotiorum and B. cinerea, and resulting transgenic plants have no adverse effect on other agronomic traits. Thus, our findings provide a novel mechanism by which a mycoparasite inhibits fungal pathogens and an environmentally friendly disease management strategy.

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of coevolution with cereal immune receptors.

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLRs) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7, and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22 and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the RNase T1/F1 family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1 family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

Suppression of ETI by PTI priming to balance plant growth and defense through an MPK3/MPK6-WRKYs-PP2Cs module.

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are required for host defense against pathogens. Although PTI and ETI are intimately connected, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that flg22 priming attenuates Pseudomonas syringae pv. tomato DC3000 (Pst) AvrRpt2-induced hypersensitive cell death, resistance, and biomass reduction in Arabidopsis. Mitogen-activated protein kinases (MAPKs) are key signaling regulators of PTI and ETI. The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression (PES). We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18, which regulates the expression of AP2C1 and PP2C5, two genes encoding protein phosphatases. Furthermore, we observed that the PTI-suppressed ETI-triggered cell death, MAPK activation, and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants. Taken together, our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

Dissecting the cotranscriptome landscape of plants and their microbiota.

Interactions between plants and neighboring microbial species are fundamental elements that collectively determine the structure and function of the plant microbiota. However, the molecular basis of such interactions is poorly characterized. Here, we colonize Arabidopsis leaves with nine plant-associated bacteria from all major phyla of the plant microbiota and profile cotranscriptomes of plants and bacteria six hours after inoculation. We detect both common and distinct cotranscriptome signatures among plant-commensal pairs. In planta responses of commensals are similar to those of a disarmed pathogen characterized by the suppression of genes involved in general metabolism in contrast to a virulent pathogen. We identify genes that are enriched in the genome of plant-associated bacteria and induced in planta, which may be instrumental for bacterial adaptation to the host environment and niche separation. This study provides insights into how plants discriminate among bacterial strains and lays the foundation for in-depth mechanistic dissection of plant-microbiota interactions.

Inter-organismal phytohormone networks in plant-microbe interactions.

Phytohormones are produced by plants and play central roles in interactions with pathogenic and beneficial microbes as well as plant growth and development. Each phytohormone pathway consists of its biosynthesis, transport, perception, and signaling and is intertwined with each other at various levels to form phytohormone networks in plants. Different kinds of microbes also produce phytohormones that exert physiological roles within microbes and manipulate phytohormone networks in plants by using phytohormones, their mimics, and proteinaceous effectors. In turn, plant-derived phytohormones can directly or indirectly through plant signaling networks affect microbial metabolism and community assembly. Therefore, phytohormone networks in plants and microbes are connected through plant and microbial phytohormones and other molecules to form inter-organismal phytohormone networks. In this review, we summarize recent progress on molecular mechanisms of inter-organismal phytohormone networks and discuss future steps necessary for advancing our understanding of phytohormone networks.

Focus on the Role of the Abiotic Environment on Interactions Between Plants and Microbes.

Interactions between plants and microbes are shaped by the physical world that surrounds them. In nature, the abiotic environment is complex, and factors such as nutrient and water availability, humidity, wind, carbon dioxide levels, salt, pollutants, and temperature all affect the growth and physiology of plants and microbes as well as their interactions. Much of our mechanistic understanding of plant-microbe interactions comes from experiments done in carefully controlled conditions. This Focus Issue looks at how aspects of the abiotic environment affect these plant-microbe interactions, and, conversely, how plant-microbe interactions affect host response to abiotic stress.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.Additional content is available on the Focus on the Role of the Abiotic Environment on Interactions Between Plants and Microbes.Complete Genome Sequence of Curtobacterium sp. C1, a Beneficial Endophyte with the Potential for In-Plant Salinity Stress AlleviationProteasomal Degradation of JAZ9 by Salt- and Drought-Induced Ring Finger 1 During Pathogen Infection.

Salicylic acid and jasmonic acid crosstalk in plant immunity.

The phytohormones salicylic acid (SA) and jasmonic acid (JA) are major players in plant immunity. Numerous studies have provided evidence that SA- and JA-mediated signaling interact with each other (SA-JA crosstalk) to orchestrate plant immune responses against pathogens. At the same time, SA-JA crosstalk is often exploited by pathogens to promote their virulence. In this review, we summarize our current knowledge of molecular mechanisms for and modulations of SA-JA crosstalk during pathogen infection.

Overexpression of NDR1 leads to pathogen resistance at elevated temperatures.

Abiotic and biotic environments influence a myriad of plant-related processes, including growth, development, and the establishment and maintenance of interaction(s) with microbes. In the case of the latter, elevated temperature has been shown to be a key factor that underpins host resistance and pathogen virulence. In this study, we elucidate a role for Arabidopsis NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) by exploiting effector-triggered immunity to define the regulation of plant host immunity in response to both pathogen infection and elevated temperature. We generated time-series RNA sequencing data of WT Col-0, an NDR1 overexpression line, and ndr1 and ics1-2 mutant plants under elevated temperature. Not surprisingly, the NDR1-overexpression line showed genotype-specific gene expression changes related to defense response and immune system function. The results described herein support a role for NDR1 in maintaining cell signaling during simultaneous exposure to elevated temperature and avirulent pathogen stressors.

Evolutionary footprint of plant immunity.

There are pieces of evidence from genomic footprints and fossil records indicating that plants have co-evolved with microbes after terrestrialization for more than 407 million years. Therefore, to truly comprehend plant evolution, we need to understand the co-evolutionary process and history between plants and microbes. Recent developments in genomes and transcriptomes of a vast number of plant species as well as microbes have greatly expanded our knowledge of the evolution of the plant immune system. In this review, we summarize recent advances in the co-evolution between plants and microbes with emphasis on the plant side and point out future research needed for understanding plant-microbial co-evolution. Knowledge of the evolution and variation of the plant immune system will better equip us on designing crops with boosted performance in agricultural fields.

Plant-Microbiota Interactions in Abiotic Stress Environments.

Abiotic stress adversely affects cellular homeostasis and ultimately impairs plant growth, posing a serious threat to agriculture. Climate change modeling predicts increasing occurrences of abiotic stresses such as drought and extreme temperature, resulting in decreasing the yields of major crops such as rice, wheat, and maize, which endangers food security for human populations. Plants are associated with diverse and taxonomically structured microbial communities that are called the plant microbiota. Plant microbiota often assist plant growth and abiotic stress tolerance by providing water and nutrients to plants and modulating plant metabolism and physiology and, thus, offer the potential to increase crop production under abiotic stress. In this review, we summarize recent progress on how abiotic stress affects plants, microbiota, plant-microbe interactions, and microbe-microbe interactions, and how microbes affect plant metabolism and physiology under abiotic stress conditions, with a focus on drought, salt, and temperature stress. We also discuss important steps to utilize plant microbiota in agriculture under abiotic stress.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An Efficient Method for DNA Purification-Free PCR from Plant Tissue.

Amplification of genomic DNA fragments by PCR is necessary for plant molecular biology approaches such as genotyping. While this is a routine molecular technique in a modern laboratory, there are still significant hurdles when analyzing a large number of samples or collecting and storing samples while in the field. Because PCR amplification directly from plant tissue is often unsuccessful due to various inhibitors, genomic DNA purification is usually required, which involves laborious and time-consuming procedures or costly materials, particularly when using commercial kits. These undermine scalability and use in less-equipped settings. In addition, plant tissues and purified DNA need to be stored under proper conditions to avoid degradation. Here, we describe a low-cost, high-throughput PCR method to amplify genomic DNA fragments from plant tissue pounded to cellulose-based filter paper without the need for DNA purification or special equipment for sample storage. In this protocol, a small punch of plant tissue is pounded to a commercially available or homemade DNA storage card and directly placed into a PCR mixture containing Tween-20, a non-ionic detergent, directly followed by PCR. We also describe the steps to prepare a homemade DNA storage card, which is easy to make and can be stored with plant tissue at room temperature for a long time without any special equipment, allowing us to test the same sample multiple times. We have used this method in at least eleven plant species, including arabidopsis, tomato, soybean, potato, cotton, and rice. Altogether, our method decreases labor and cost, thereby increasing throughput and making plant DNA-based molecular diagnostic assays accessible to resource-limited settings, including classrooms, and facilitating sample collection in the field. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Making a homemade cellulose-based DNA storage card Basic Protocol 2: Pounding plant tissue on a DNA storage card Basic Protocol 3: DNA-purification free PCR.