Response to Persistent ER Stress in Plants: a Multiphasic Process that Transitions Cells from Prosurvival Activities to Cell Death

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1\u27s activity declined as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amidst a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death

Heterosis

Heterosis refers to the phenomenon that progeny of diverse varieties of a species or crosses between species exhibit greater biomass, speed of development, and fertility than both parents. Various models have been posited to explain heterosis, including dominance, overdominance, and pseudo-overdominance. In this Perspective, we consider that it might be useful to the field to abandon these terms that by their nature constrain data interpretation and instead attempt a progression to a quantitative genetic framework involving interactions in hierarchical networks. While we do not provide a comprehensive model to explain the phenomenology of heterosis, we provide the details of what needs to be explained and a direction of pursuit that we feel should be fruitful

polishCLR: a Nextflow workflow for polishing PacBio CLR genome assemblies

Long-read sequencing has revolutionized genome assembly, yielding highly contiguous, chromosome-level contigs. However, assemblies from some third generation long read technologies, such as Pacific Biosciences (PacBio) Continuous Long Reads (CLR), have a high error rate. Such errors can be corrected with short reads through a process called polishing. Although best practices for polishing non-model de novo genome assemblies were recently described by the Vertebrate Genome Project (VGP) Assembly community, there is a need for a publicly available, reproducible workflow that can be easily implemented and run on a conventional high performance computing environment. Here, we describe polishCLR (https://github.com/isugifNF/polishCLR), a reproducible Nextflow workflow that implements best practices for polishing assemblies made from CLR data. PolishCLR can be initiated from several input options that extend best practices to suboptimal cases. It also provides re-entry points throughout several key processes including identifying duplicate haplotypes in purge_dups, allowing a break for scaffolding if data are available, and throughout multiple rounds of polishing and evaluation with Arrow and FreeBayes. PolishCLR is containerized and publicly available for the greater assembly community as a tool to complete assemblies from existing, error-prone long-read data.This is a pre-print of the article Chang, Jennifer, Amanda R. Stahlke, Sivanandan Chudalayandi, Benjamin D. Rosen, Anna K. Childers, and Andrew Severin. "polishCLR: a Nextflow workflow for polishing PacBio CLR genome assemblies." bioRxiv (2022). DOI: 10.1101/2022.02.10.480011. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted

A repackaged CRISPR platform increases homology-directed repair for yeast engineering

Inefficient homology-directed repair (HDR) constrains CRISPR–Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform—lowered indel nuclease system enabling accurate repair (LINEAR)—which enhanced HDR rates (to 67–100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.This is a post-peer-review, pre-copyedit version of an article published in Nature Chemical Biology. The final authenticated version is available online at DOI: 10.1038/s41589-021-00893-5. Copyright © 2021, The Author(s). Posted with permission

Suppl dataset 3 ATACseq

ATACseq analysis was conducted in triplicate to identify changes in chromatin “openness” or accessibility in response to ER stress. Maize B73 seedlings were treated with 5 g ml-1 TM for 0, 6 and 12 h. Nuclei were isolated from root samples and incubated with Tn5 transposase to tag DNA fragments in regions of open chromatin. The tagged DNA was PCR amplified with primers directed against the Tn5 inserts and used to construct a cDNA library, which was subject to DNA sequencing. Short read sequences were aligned to the maize genome version 4, and peaks were called for each replicate at every time point. Differential peak scores representing changes in promoter openness in the 500 or 5000 bp upstream region for each gene for 6 h vs 0 h and 12 h vs 0 h were then calculated. Tab 1: from the start of transcription to 500 bp upstream. Tab 2: from the start of transcription to 5000 bp upstream

Suppl dataset 2 CHIPseq bZIP60 & 17

Chromatin immunoprecipitation analysis was carried out in triplicate to identify the direct transcriptional targets for ZmbZIP60 and ZmbZIP17. GFP tagged forms of the bZIP transcription factors (minus their transmembrane and lumen-facing domains) were transfected into maize seedling mesophyll protoplasts and after incubation nuclei were isolated, chromatin extracted and fragmented by sonication. GFP tagged ZmbZIP60 and ZmbZIP91 were immunoprecipitated with anti-GFP antibody and the coimmunoprecipitated DNA fragments were eluted, used to construct DNA libraries and subject to DNA sequencing. Read sequences were aligned to maize genome version 4. Peaks were identified and their intersection with 500 bp upstream in the promoter regions of the various genes was computed to give a peak score

Response to Persistent ER Stress in Plants: a Multiphasic Process that Transitions Cells from Prosurvival Activities to Cell Death

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of the promiscuous ribonuclease activity of ZmIRE1 that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 mRNA to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1's activity declined as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amidst a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death.This is a manuscript of an article published as Srivastava, Renu, Zhaoxia Li, Giulia Russo, Jie Tang, Ran Bi, Usha Muppirala, Sivanandan Chudalayandi, Andrew Severin, Mingze He, Samuel I. Vaitkevicius, Carolyn J. Lawrence-Dill, Peng Liu, Ann E. Stapleton, Diane C. Bassham, Federica Brandizzi, and Stephen H. Howell. "Response to Persistent ER Stress in Plants: A Multiphasic Process That Transitions Cells from Prosurvival Activities to Cell Death." The Plant Cell (2018): tpc-00153. DOI: 10.1105/tpc.18.00153. Posted with permission.</p

Data from: Persistent ER stress in plants: a multiphasic process that transitions cells from prosurvival activities to cell death

The unfolded protein response (UPR) is a highly conserved response that protects plants from adverse environmental conditions. The UPR is elicited by endoplasmic reticulum (ER) stress, in which unfolded and misfolded proteins accumulate within the ER. Here, we induced the UPR in maize (Zea mays) seedlings to characterize the molecular events that occur over time during persistent ER stress. We found that a multiphasic program of gene expression was interwoven among other cellular events, including the induction of autophagy. One of the earliest phases involved the degradation by regulated IRE1-dependent RNA degradation (RIDD) of RNA transcripts derived from a family of peroxidase genes. RIDD resulted from the activation of ZmIRE1 for promiscuous ribonuclease activity that attacks the mRNAs of secreted proteins. This was followed by an upsurge in expression of the canonical UPR genes indirectly driven by ZmIRE1 due to its splicing of Zmbzip60 to make an active transcription factor that directly upregulates many of the UPR genes. At the peak of UPR gene expression, a global wave of alternative RNA processing led to the production of many aberrant UPR gene transcripts, likely tempering the ER stress response. During later stages of ER stress, ZmIRE1’s activity declined as did the expression of survival modulating genes, Bax inhibitor1 and Bcl-2-associated athanogene7, amidst a rising tide of cell death. Thus, in response to persistent ER stress, maize seedlings embark on a course of gene expression and cellular events progressing from adaptive responses to cell death