A seed resource for screening functionally redundant genes and isolation of new mutants impaired in CO₂ and ABA responses

Abstract

The identification of homologous genes with functional overlap in forward genetic screens is $)! severely limited. Here we report the generation of over 14,000 amiRNA-expressing plants that $*! enable screens of the functionally redundant gene space in Arabidopsis. A protocol is developed %+! here for isolating robust and reproducible amiRNA-mutants. Examples of validation approaches %"! and essential controls are presented for two new amiRNA mutants that exhibit genetically %#! redundant phenotypes and circumvent double mutant lethality. In a forward genetic screen for %$! abscisic acid (ABA)-mediated inhibition of seed germination, amiRNAs that target combinations %%! of known redundant ABA receptor and SnRK2 kinase genes were rapidly isolated, providing a %&! strong proof of principle for this approach. A new ABA insensitive amiRNA line is isolated, which %'! targets three genes encoding avirulence-induced gene2-like (AIG2) genes. A thermal imaging %(! screen for plants with impaired stomatal opening in response to low CO 2 exposure led here to %)

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses

“…Genomic DNA for candidate amiRNA lines was extracted (Kang, Cho, Yoon, & Eun, 1998) and used as templates, and the primers (primers pha2804f and pha3479r; Hauser et al, 2019); Table S1) were used to amplify the PCR product. The purified PCR samples were sent for sequencing to identify the amiRNA present (Hauser et al, 2019). The genes targeted by the amiRNA could be putatively identified by using the ‘Target Search Function’ on the WMD3 website (Ossowski, Fitz, Schwab, Riester, & Weigel, 2010).…”

“…The genes targeted by the amiRNA could be putatively identified by using the ‘Target Search Function’ on the WMD3 website (Ossowski, Fitz, Schwab, Riester, & Weigel, 2010). Several studies have demonstrated the accuracy of in‐silico predictions of amiRNA target genes (Hauser et al, 2013, 2019; Takahashi et al, 2020; Zhang et al, 2018).…”

“…In previous research, we computationally designed artificial microRNAs (amiRNAs; Schwab, Ossowski, Riester, Warthmann, & Weigel, 2006), and synthesized 22,000 amiRNAs designed to combinatorially co‐silence closely homologous gene clade members (Hauser et al, 2013). These amiRNAs were separated into 10 amiRNA libraries for genome‐wide knockdown of homologous gene family members based on the predicted functional classes of the genes they target (Hauser et al, 2013; Hauser et al, 2019). Using these 22,000 amiRNAs, libraries were synthesized (Hauser et al, 2013) and used to transform Arabidopsis .…”

“…Using these 22,000 amiRNAs, libraries were synthesized (Hauser et al, 2013) and used to transform Arabidopsis . To date, 14,000 T2 generation lines have been generated (Hauser et al, 2019). Recent studies have used this resource to discover new genes and determine their functions in plant hormone signalling and transport, illustrating the power of this system (Hauser et al, 2013, 2019; Takahashi et al, 2020; Zhang et al, 2018).…”

A role for calcium‐dependent protein kinases in differential CO2‐ and ABA‐controlled stomatal closing and low CO2‐induced stomatal opening in Arabidopsis

“…After stabilization of gs for 45–60 min, stomatal conductance of individual intact leaves was measured for 10 min in the dark, followed by red light (600 μmol m −2 s −1 ) exposure for 30 min. After red light treatment, blue light (10 μmol m −2 s −1 ) was applied and superimposed on red light, and stomatal conductance was measured for another 30 min as described previously (Shimazaki et al ., 2007; Hauser et al ., 2019).…”