Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

Abstract

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to the hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.

PcGCE is a potent elicitor of defense responses in aspen

“…We also identified many transcription factors, in particular, several WRKY factors. Some of these genes were found similarly affected in Arabidopsis stems ectopically overexpressing PcGCE (Tsai et al, 2017), as shown in Table S9. The identified genes constitute transcriptional modules that likely operate in recognition of PcGCE and other molecular patterns associated with pathogens to activate defense systems in aspen.…”

“…Similar to aspen, Arabidopsis expressing PcGCE exhibited transcriptomic changes typical of biotic stress responses, SA signaling (Tsai et al, 2017). Moreover, several biotic-stress related genes were affected in common in both species, including glutamate receptor GLR2.7, recently shown to constitute a core stress response gene (Bjornson et al, 2021), WRKY51 that mediates JA signaling inhibition by SA and low oleic acid levels (Gao et al, 2011), SOBIR1 that together with BAK1 and RLK23 recognizes NLP20 peptide inducing necrosis (Albert et al, 2015), and other genes which we identified as putative candidates for early PcGCE responses in aspen leaves (Table S9).…”

“…However, microbial enzymes expressed in planta to modify plant cell wall sometimes triggered diverse immune responses, such as premature leaf senescence, necrotic lesions in the leaves, accumulation of reactive oxygen species (ROS) and induction of genes involved in biotic and abiotic stress responses (Bailey et al, 1990;Avni et al, 1994;de Buanafina 2012;Pogorelko et al, 2013;Tsai et al, 2017). It has been suggested that cell wall modifying enzymes could generate oligosaccharide fractions such as, oligogalacturonides (OGs) (Legendre et al, 1993;Norman et al, 1999;D'Ovidio et al, 2004), cellooligomers (Souza et al, 2017) or xyloglucan oligosaccharides (Claverie et al, 2018), which can be perceived by the plant cell as damage-associated molecular patterns (DAMPs).…”

“…When GCE from necrotrophic wood decaying white-rot basidiomycete, Phanerochaete carnosa Burt (PcGCE), was ectopically expressed in Arabidopsis thaliana (L.) Heynh. and hybrid aspen, leaf senescence was accelerated and growth was reduced (Tsai et al, 2012;Gandla et al, 2015;Tsai et al, 2017).…”

Post-Synthetic Reduction of Pectin Methylesterification Causes Morphological Abnormalities and Alterations to Stress Response in Arabidopsis thaliana

“…However, due to the complexities of cell wall structure and signaling, it is difficult to pinpoint how pectin exerts control over these functions. Previous research has shown that constitutive expression of microbial hydrolases in the plant apoplast is a useful approach to investigate the impacts of cell wall post-synthetic modification on cell wall signaling, plant fitness, and stress responses [ 15 , 57 , 58 , 59 , 60 , 61 ]. Since plant growth is often halted under stress, this approach provides an opportunity to understand pectin’s contribution to both plant cell wall mechanics and plant response to stresses.…”