Imaging-Based Resistance Assay Using Enhanced Luminescence-Tagged Pseudomonas syringae Reveals a Complex Epigenetic Network in Plant Defense Signaling Pathways.

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the Nanoluc luciferase (NL) from the deep-sea shrimp Oplophorus gracilirostris, a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was more than 100 times brighter than P. syringae tagged with the luxCDABE operon from Photorhabdus luminescens, one of the existing luciferase-based strains. In planta imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, more than 30 epigenetic mutants were analyzed for their resistance traits because the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pseudomonas syringae pv. tomato. Dicer-like and Argonaute proteins, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for effector-triggered immunity and basal resistance to P. syringae pv. tomato. In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge about complex defense signaling pathways.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis.

To assess the role of MORC1 in epigenetics in relation to plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato-inoculated wild type (WT) Arabidopsis, the morc1/2 double mutant, or both. Most changes in chromatin accessibility, scored by DNase I hypersensitive sites (DHSs), were located in the promoters of genes and transposable elements (TEs). Comparisons between morc1/2 and WT receiving the same treatment revealed differential DHSs (dDHSs) predominantly associated with heterochromatic TEs. By contrast, comparisons between mock- and P. syringae pv. tomato-inoculated plants from the same genotype showed dDHSs associated with biotic and abiotic stress-related genes; a smaller but significant population was in TEs. Moreover, many defense genes, including PR-1, PR-2, and PR-5, were proximal to P. syringae pv. tomato-induced, TE-associated dDHSs. A random subset of these defense genes showed moderately delayed or reduced expression or both in P. syringae pv. tomato-infected morc1/2 as compared with WT. MORC1 was physically bound to chromatin in a P. syringae pv. tomato infection-responsive manner at sites dispersed throughout the genome. Notably, silencing of TE-associated dDHSs proximal to these infection-induced, MORC1-interacting sites led to significant suppression of P. syringae pv. tomato-induced transcription of adjacent defense genes, including PR-1. These results provide evidence that MORC1 is associated with TEs and suggest that a subset of these TEs may help regulate their proximal defense genes.

The Arabidopsis immune adaptor SRFR1 interacts with TCP transcription factors that redundantly contribute to effector-triggered immunity.

The plant immune system must be tightly controlled both positively and negatively to maintain normal plant growth and health. We previously identified SUPPRESSOR OF rps4-RLD1 (SRFR1) as a negative regulator specifically of effector-triggered immunity. SRFR1 is localized in both a cytoplasmic microsomal compartment and in the nucleus. Its TPR domain has sequence similarity to TPR domains of transcriptional repressors in other organisms, suggesting that SRFR1 may negatively regulate effector-triggered immunity via transcriptional control. We show here that excluding SRFR1 from the nucleus prevented complementation of the srfr1 phenotype. To identify transcription factors that interact with SRFR1, we screened an Arabidopsis transcription factor prey library by yeast two-hybrid assay and isolated six class I members of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factor family. Specific interactions were verified in planta. Although single or double T-DNA mutant tcp8, tcp14 or tcp15 lines were not more susceptible to bacteria expressing AvrRps4, the triple tcp8 tcp14 tcp15 mutant displayed decreased effector-triggered immunity mediated by the resistance genes RPS2, RPS4, RPS6 and RPM1. In addition, expression of PATHOGENESIS-RELATED PROTEIN2 was attenuated in srfr1-4 tcp8-1 tcp14-5 tcp15-3 plants compared to srfr1-4 plants. To date, TCP transcription factors have been implicated mostly in developmental processes. Our data indicate that one function of a subset of TCP proteins is to regulate defense gene expression in antagonism to SRFR1, and suggest a mechanism for an intimate connection between plant development and immunity.

Co-immunoprecipitation for Assessing Protein-Protein Interactions in Agrobacterium-Mediated Transient Expression System in Nicotiana benthamiana.

The characterization of protein-protein interactions (PPI) often provides functional information about a target protein. Yeast-two-hybrid (Y2H) and luminescence/fluorescence-based detections, therefore, have been widely utilized for assessing PPI. In addition, a co-immunoprecipitation (co-IP) method has also been adopted with transient protein expression in Nicotiana benthamiana (N. benthamiana) infiltrated with Agrobacterium tumefaciens. Herein, we describe a co-IP procedure in which structural maintenance of chromosome 1 (SMC1), identified from a Y2H screening, was verified as an interacting partner for microchidia 1 (MORC1), a protein well known for its function in plant immunity and epigenetics. SMC1 and MORC1 were transiently expressed in N. benthamiana when infiltrated by Agrobacterium with the respective genes. From this approach, we identified a region of SMC1 responsible for interacting with MORC1. The co-IP method, of which outputs are mainly from immunoblot analysis, provided information about target protein expression as well, which is often useful for troubleshooting. Using this feature, we showcased a PPI confirmation from our SMC1-MORC1 study in which a full-length SMC1 protein was not detectable, and, therefore, a subsequent truncated mutant analysis had to be employed for PPI verification.

The Arabidopsis resistance-like gene SNC1 is activated by mutations in SRFR1 and contributes to resistance to the bacterial effector AvrRps4.

The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1.

Constitutive expression of a pea apyrase, psNTP9, increases seed yield in field-grown soybean.

To address the demand for food by a rapidly growing human population, agricultural scientists have carried out both plant breeding and genetic engineering research. Previously, we reported that the constitutive expression of a pea apyrase (Nucleoside triphosphate, diphosphohydrolase) gene, psNTP9, under the control of the CaMV35S promoter, resulted in soybean plants with an expanded root system architecture, enhanced drought resistance and increased seed yield when they are grown in greenhouses under controlled conditions. Here, we report that psNTP9-expressing soybean lines also show significantly enhanced seed yields when grown in multiple different field conditions at multiple field sites, including when the gene is introgressed into elite germplasm. The transgenic lines have higher leaf chlorophyll and soluble protein contents and decreased stomatal density and cuticle permeability, traits that increase water use efficiency and likely contribute to the increased seed yields of field-grown plants. These altered properties are explained, in part, by genome-wide gene expression changes induced by the transgene.

High-Throughput Targeted Transcriptional Profiling of Defense Genes Using RNA-Mediated Oligonucleotide Annealing, Selection, and Ligation with Next-Generation Sequencing in Arabidopsis.

Tracking RNA transcription has been one of the most powerful tools to gain insight into the biological process. While a wide range of molecular methods such as northern blotting, RNA-seq, and quantitative RT-PCR are available, one of the barriers in transcript analysis is an inability to accommodate a sufficient number of samples to achieve high resolution in dynamic transcriptional changes. RASL-seq (RNA-mediated oligonucleotide Annealing, Selection, and Ligation with next-generation sequencing) is a sequencing-based transcription profiling tool that processes hundreds of samples assessing a set of over a hundred genes with a fraction of the cost of a conventional RNA-seq. We described a RASL-seq protocol for assessing 288 genes mostly including defense genes to capture their dynamic nature. We demonstrated that this transcriptional profiling method produced a highly reliable outcome comparable to a conventional RNA-seq and quantitative RT-PCR.