Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Image-Based Quantification of Arabidopsis thaliana Stomatal Aperture from Leaf Images.

The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from bright-field microscopy images containing mesophyll tissue as noisy backgrounds. By combining a You Only Look Once X-based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved a mean average precision with an intersection of union (IoU) threshold of 50% value of 0.875 (stomata detection performance) and an IoU of 0.745 (pore segmentation performance) against images of leaf discs taken with a bright-field microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots.

Chitin-induced systemic disease resistance in rice requires both OsCERK1 and OsCEBiP and is mediated via perturbation of cell-wall biogenesis in leaves.

Chitin is a well-known elicitor of disease resistance and its recognition by plants is crucial to perceive fungal infections. Chitin can induce both a local immune response and a systemic disease resistance when provided as a supplement in soils. Unlike local immune responses, it is poorly explored how chitin-induced systemic disease resistance is developed. In this study, we report the systemic induction of disease resistance against the fungal pathogen Bipolaris oryzae by chitin supplementation of soils in rice. The transcriptome analysis uncovered genes related to cell-wall biogenesis, cytokinin signaling, regulation of phosphorylation, and defence priming in the development of chitin-induced systemic response. Alterations of cell-wall composition were observed in leaves of rice plants grown in chitin-supplemented soils, and the disease resistance against B. oryzae was increased in rice leaves treated with a cellulose biosynthesis inhibitor. The disruption of genes for lysin motif (LysM)-containing chitin receptors, OsCERK1 (Chitin elicitor receptor kinase 1) and OsCEBiP (Chitin elicitor-binding protein), compromised chitin-induced systemic disease resistance against B. oryzae and differential expression of chitin-induced genes found in wild-type rice plants. These findings suggest that chitin-induced systemic disease resistance in rice is caused by a perturbation of cell-wall biogenesis in leaves through long-distance signalling after local recognition of chitins by OsCERK1 and OsCEBiP.

Disease resistance and growth promotion activities of chitin/cellulose nanofiber from spent mushroom substrate to plant.

Some studies have reported the method for treating the spent mushroom substrate (SMS). However, the effective use as a functional raw material based on properties of SMS remains a formidable challenge. In this study, we investigated the usefulness of SMS in agriculture to develop a new method for treating and utilizing it. First, we attempted to isolate chitin/cellulose nanofiber complex (CCNFC) from SMS using chemical pretreatment and mechanical fibrillation. The characterization results like SEM, FT-IR, and XRD showed that we successfully isolated the CCNFC from SMS. Second, we explored the biological activities of the CCNFC for its potential application as a functional agricultural nanomaterial. CCNFC water dispersion with low concentration (0.1 and 1 mg/mL) exhibited significant plant disease resistance and plant growth promotion activities. Our results suggested that SMS may provide a useful source of functional agricultural nanomaterial, which may contribute to treating and applying it in agriculture.

Simultaneous mutations in SMN1 and SUMM2 fully suppress the dwarf and autoimmune phenotypes of Arabidopsis mpk4 mutant.

Disruption of the Arabidopsis mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4 (hereafter designated as MEKK1 pathway), leads to the activation of distinct NLRs (nucleotide-binding and leucine-rich repeat receptors), TNL (TIR-type NLR) SMN1, and CNL (CC-type NLR) SUMM2, resulting in dwarf and autoimmune phenotypes. Unlike mekk1 and mkk1mkk2 mutants, the dwarf and autoimmune phenotypes of mpk4 are only partially suppressed by the summ2 mutation, suggesting a significant contribution of SMN1 to the mpk4 phenotypes. However, full suppression of mpk4 by the smn1summ2 double mutation remains to be elucidated. To address this key question, we generated a mpk4smn1summ2 triple mutant and analyzed the dwarf and constitutive cell death phenotypes. The mpk4smn1summ2 triple mutant showed restoration of plant size with no detectable cell death, indicating full suppression of the dwarf and autoimmune phenotypes. These results suggest that SMN1 and SUMM2 constitute a robust surveillance system for the MEKK1 pathway against pathogen infection.

Arabidopsis SMN2/HEN2, Encoding DEAD-Box RNA Helicase, Governs Proper Expression of the Resistance Gene SMN1/RPS6 and Is Involved in Dwarf, Autoimmune Phenotypes of mekk1 and mpk4 Mutants.

In Arabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4, is important for basal resistance and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants, smn2, had defects in the SMN2 gene, encoding a DEAD-box RNA helicase. SMN2 is identical to HEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control and RNA processing. Aberrant SMN1/RPS6 transcripts were detected in smn2 and hen2 mutants. Disease resistance against Pseudomonas syringae pv. tomato DC3000 (hopA1), which is conferred by SMN1/RPS6, was decreased in smn2 mutants, suggesting a functional connection between SMN1/RPS6 and SMN2/HEN2. We produced double mutants mekk1smn2 and mpk4smn2 to determine whether the smn2 mutations suppress the dwarf, autoimmune phenotypes of the mekk1 and mpk4 mutants, as the smn1 mutations do. As expected, the mekk1 and mpk4 phenotypes were suppressed by the smn2 mutations. These results suggested that SMN2 is involved in the proper function of SMN1/RPS6. The Gene Ontology enrichment analysis using RNA-seq data showed that defense genes were downregulated in smn2, suggesting a positive contribution of SMN2 to the genome-wide expression of defense genes. In conclusion, this study provides novel insight into plant immunity via SMN2/HEN2, an essential component of the nuclear RNA exosome.

Disruption of the MAMP-Induced MEKK1-MKK1/MKK2-MPK4 Pathway Activates the TNL Immune Receptor SMN1/RPS6.

Mitogen-activated protein kinase (MAPK) pathways have a pivotal role in innate immunity signaling in plants. In Arabidopsis, the MAPK pathway that consists of MEKK1, MKK1/MKK2 and MPK4 is involved in pattern-triggered immunity signaling upstream of defense gene expression. This pathway is partly guarded by SUMM2, a nucleotide-binding domain leucine-rich repeat (NLR) protein, which is activated by disruption of the MAPK pathway. To identify other components required for the guard mechanism, here we developed a new mutant screening system utilizing a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Mutants with suppression of the dwarf, autoimmune phenotypes were identified, and one locus responsible for the phenotype was designated as suppressor of MEKK1N overexpression-induced dwarf 1 (SMN1). MutMap analysis revealed that SMN1 encodes the Toll/Interleukin-1 receptor (TIR)-class NLR protein RPS6, a previously identified resistant protein against bacterial pathogen Pseudomonas syringae pv. tomato expressing the HopA1 effector. Importantly, mutations in SMN1/RPS6 also partially suppressed the dwarf, autoimmune phenotypes of mekk1 and mpk4 plants. Our results suggest that the two structurally distinct NLR proteins, SMN1/RPS6 and SUMM2, monitor integrity of the MEKK1-MKK1/MKK2-MPK4 pathway.