High‐resolution expression profiling of selected gene sets during plant immune activation

Abstract

The plant immune system involves detection of pathogens via both cell-surface and intracellular receptors. Both receptor classes can induce transcriptional reprogramming that elevates disease resistance. To assess differential gene expression during plant immunity, we developed and deployed quantitative sequence capture (CAP-I). We designed and synthesized biotinylated single-strand RNA bait libraries targeted to a subset of defense genes, and generated sequence capture data from 99 RNA-seq libraries. We built a data processing pipeline to quantify the RNA-CAP-I-seq data, and visualize differential gene expression. Sequence capture in combination with quantitative RNA-seq enabled cost-effective assessment of the expression profile of a specified subset of genes. Quantitative sequence capture is not limited to RNA-seq or any specific organism and can potentially be incorporated into automated platforms for high-throughput sequencing.1610

Chromatin accessibility landscapes activated by cell surface and intracellular immune receptors

“…Infiltration of Pf0-1:AvrRps4 mut activates SRMI, and Pf0-1:AvrRps4 WT activates ‘SRMI+IRMI’ (Supplementary Fig. 5a), as in previous reports 4,8 . We took samples at 4 hours post-infiltration (hpi) for ATAC-seq to monitor early changes during immune activation (Supplementary Fig.…”

“…We study the Arabidopsis RPS4/RRS1 NLR pair that detects bacterial effectors AvrRps4 and PopP2. Using a Pseudomonas strain that solely delivers one of these effectors, we defined early RPS4/RRS1-dependent transcriptional responses in Arabidopsis leaves 6,7 , and showed that 4 hours after infiltration, SRMI together with IRMI (‘SRMI+IRMI’) elevates the expression of defense-related genes more strongly compared to SRMI alone 6,8 . This early timepoint precedes accumulation of defense hormone salicylic acid (SA) and gene reprogramming in response to increased endogenous SA level 6,8 .…”

“…Using a Pseudomonas strain that solely delivers one of these effectors, we defined early RPS4/RRS1-dependent transcriptional responses in Arabidopsis leaves 6,7 , and showed that 4 hours after infiltration, SRMI together with IRMI (‘SRMI+IRMI’) elevates the expression of defense-related genes more strongly compared to SRMI alone 6,8 . This early timepoint precedes accumulation of defense hormone salicylic acid (SA) and gene reprogramming in response to increased endogenous SA level 6,8 . This implies that IRMI-enhanced transcriptional regulation plays an essential role in conferring robust immune responses against pathogens 4 , but how IRMI activates defense genes remains unclear.…”

“…Activation of SRMI, IRMI and ‘SRMI+IRMI’ lead to rapid transcriptional reprogramming 6,8,10 . Many transcriptional regulatory components are involved in orchestrating effective immunity in plants 11,12 , notably transcription factors (TFs) 13 , transcription co-repressors 14 , the Mediator complex 15 , histone-modifying enzymes 16 , and histone remodellers 17 .…”

Targeted RNA sequencing enhances gene expression profiling of ultra-low input samples

“…The use of targeted RNA-seq (CaptureSeq) can help resolve this issue by focusing sequencing on genes of interest and allowing the interrogation of low-expressed genes and transcripts that would be missed by conventional methods [2][3][4][5]. Targeted RNA-seq is a widely applicable method and has been successfully utilized to enrich genes and transcripts from mammals [2,3], plants [6], fungi [7], bacteria [8] and viruses [9]. CaptureSeq performs well on different sample types, including formalin-fixed paraffin-embedded (FFPE) samples with highly degraded RNA and has been widely applied to identify molecular alterations in cancer in both research and clinical settings [10][11][12][13][14][15].…”