Probing natural variation of IRE1 expression and endoplasmic reticulum stress responses in Arabidopsis accessions

Abstract

The environmental effects shape genetic changes in the individuals within plant populations, which in turn contribute to the enhanced genetic diversity of the population as a whole. Thus, individuals within the same species can acquire and accumulate genetic differences in their genomes depending on their local environment and evolutionary history. IRE1 is a universal endoplasmic reticulum (ER) stress sensor that activates an evolutionarily conserved signalling cascade in response to biotic and abiotic stresses. Here, we selected nine different Arabidopsis accessions along with the reference ecotype Columbia-0, based on their geographical origins and differential endogenous IRE1 expression under steady-state conditions to investigate the natural variation of ER stress responses. We cloned and analysed selected upstream regulatory regions of IRE1a and IRE1b, which revealed differential levels of their inducibility. We also subjected these accessions to an array of biotic and abiotic stresses including heat, ER stress-inducing chemical tunicamycin, phytohormone salicylic acid, and pathogen infection. We measured IRE1-mediated splicing of its evolutionarily conserved downstream client as well as transcript accumulation of ER-resident chaperones and co-chaperones. Collectively, our results illustrate the expression polymorphism of a major plant stress receptor and its relationship with molecular and physiological ER stress sensitivity.

The interplay of GTP-binding protein AGB1 with ER stress sensors IRE1a and IRE1b modulates Arabidopsis unfolded protein response and bacterial immunity

“… 40 , 63 We detected an enhanced Tm sensitivity in the ire1a-2/1b-4 seedlings, which was expected given the pivotal roles of IRE1a and IRE1b in plant ER stress responses, and is also consistent with the earlier reports. 64 , 65 The triple mutant ire1a-2/1b-4/agb1-2 displayed a statistically significant reduction in the fresh weight; however, its Tm sensitivity was not further enhanced compared to the double mutant ire1a-2/1b-4 seedlings ( Figure 2 a,b ).…”

“…The single mutant agb1-2 and double mutant ire1a-2/1b-4 displayed significantly enhanced bacterial loads compared to Col-0, which is consistent with earlier reports. 42 , 59 , 65 The triple mutant ire1a-2/1b-4/agb1-2 showed a further increased bacterial susceptibility compared to ire1a-2/1b-4 and agb1-2 , supporting 0.7 log (~5 times) more bacterial growth than Col-0 ( Figure 5 ), and further substantiating the hypothesis that the IRE1 and AGB1 likely act non-redundantly and have a cumulative contribution to plant stress responses, including immunity to a bacterial pathogen.

Figure 5.

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“…Previous studies reported the independent contributions of IRE1a/IRE1b 35 , 36 , 42 , 65 and AGB1 2 , 19 , 40 , 47 , 56 , 59 , 60 to plant immune responses. In our study, we provide evidence that both IRE1a/IRE1b and AGB1 are required for initiating the basal defense response against the virulent bacterial pathogen ( Figure 5 ), as the triple mutant ire1a-2/1b-4/agb1-2 harbored a significantly higher number of bacteria than did the ire1a-2/1b-4 and agb1-2 plants.…”

“… 67–69 Our study showed that the Arabidopsis G protein subunit β1 (AGB1) cross-talks with the IRE1a and IRE1b homologs to modulate the abiotic and biotic ER stress response mechanisms. While the functions, mechanism of action, and importance of both IRE1a/IRE1b 34–36 , 40 , 42 , 65 and AGB1 2 , 19 , 40 , 47 , 56 , 60 in Arabidopsis have been well characterized, the nature of their cooperative roles in UPR remains unclear. Our findings support the notion that AGB1 and IRE1 signaling pathways are at least partially independent and can act synergistically in their response mechanisms, as proposed in an earlier study.…”

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

“…We established a novel quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein fragments and total RNA isolated from Arabidopsis leaves to determine bZIP60us mRNA cleavage efficiency. Previously, we and others have measured Arabidopsis, maize, potato, or tomato IRE1’s RNase output in plant cells using bZIP60 mRNA splicing assays, utilizing both semi-quantitative RT-PCR agarose gel-based and quantitative, qRT-PCR-based methodologies ( Deng et al, 2011 , 2013 ; Nagashima et al, 2011 ; Moreno et al, 2012 ; Zhang et al, 2015 ; Gaguancela et al, 2016 ; Afrin et al, 2020b ; Li et al, 2020 ; Kaur and Kaitheri Kandoth, 2021 ). While useful, these techniques do not allow precise time-resolved measurements of RNA processing by IRE1, and can only capture the splicing/degradation executed by the endogenous, wild-type versions of the IRE1 proteins.…”