Nathalie Beaudoin

The phytotoxin thaxtomin A (TA) is the key pathogenicity factor synthesized by the bacteria Streptomyces scabiei, the main causal agent of common scab of potato (Solanum tuberosum L.). TA treatment of potato tuber flesh produces a brown color that was attributed to necrosis. The intensity of TA-induced browning was generally thought to correlate with potato sensitivity to the disease. In this study, we found that TA-induced browning was much more intense in the potato tuber flesh of the common scab moderately resistant variety Russet Burbank (RB) than that observed in tubers of the disease-susceptible variety Yukon Gold (YG). However, there was no significant difference in the level of TA-induced cell death detected in both varieties, suggesting that tubers response to TA does not correlate with the level of sensitivity to common scab. TA-treated potato tuber tissues accumulated significantly higher levels of phenolic compounds than untreated controls, with a higher phenol content detected in RB TA-treated tissues than in those of YG. Browning was associated with a significant induction of the expression of genes of the phenylpropanoid pathway in RB tubers, indicating that TA activated this metabolic pathway. These results suggest that tuber flesh browning induced by TA is due to the accumulation of phenolic compounds. These phenolics may play a role in the protection of potato tubers against S. scabiei.

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A‐habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label‐free quantitative proteomic analysis revealed changes in the accumulation of defence‐related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz‐type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf‐bud cuttings. S . scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.

Common scab is a potato disease characterized by the formation of scab-like lesions on the surface of potato tubers. The actinobacterium Streptomyces scabiei is the main causal agent of common scab. During infection, this bacterium synthesizes the phytotoxin thaxtomin A which is essential for the production of disease symptoms. While thaxtomin A can activate an atypical programmed cell death in plant cell suspensions, it is possible to gradually habituate plant cells to thaxtomin A to provide resistance to lethal phytotoxin concentrations. Potato ‘Russet Burbank’ calli were habituated to thaxtomin A to regenerate the somaclone RB9 that produced tubers more resistant to common scab than those obtained from the original cultivar. Compared to the Russet Burbank cultivar, somaclone RB9 generated up to 22% more marketable tubers with an infected tuber area below the 5% threshold. Enhanced resistance was maintained over at least two years of cultivation in the field. However, average size of tubers was significantly reduced in somaclone RB9 compared to the parent cultivar. Small RB9 tubers had a thicker phellem than Russet Burbank tubers, which may contribute to improving resistance to common scab. These results show that thaxtomin A-habituation in potato is efficient to produce somaclones with increased and durable resistance to common scab.

The outer potato periderm layer consists of dead suberized cells. Suberin, a protective biopolymer, is made of a polyaliphatic portion covalently linked to polyaromatic moieties. Evidence accumulates that Streptomyces scabies, the main causal agent of potato common scab, can degrade the suberin aliphatic part but its ability to degrade the aromatic portion has not been documented. This polyaromatic portion is mainly composed of cinnamic acids. In this study, two cinnamates (trans-ferulic or p-coumaric acids) were added to the culture medium of S. scabies strains EF-35 and 87.22. HPLC quantification revealed that both strains efficiently utilized these compounds. A proteomic study coupled with gene expression analysis led to the identification of putative catabolic pathways for cinnamates. Catabolism of both compounds appeared to occur via the β-ketoadipate pathway. Gene SCAB_15301, encoding for a putative vanillate monooxygenase, was partly deleted from S. scabies strain 87.22 genome. The mutant retained its ability to catabolize trans-ferulic acid into vanillate but lost its ability to further degrade the latter compound. When the wild-type mutant and complemented strains were grown in the presence of suberin-enriched potato periderm, accumulation of vanillic acid was observed only in the mutant culture medium. This work presents evidence that S. scabies can degrade not only the aliphatic part of suberin but also the constituents of suberin aromatic portion. This may provide ecological and pathological advantages to S. scabies as a saprophyte and pathogen.

Background: Thaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB). Results: The induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only. Conclusions: Addition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin’s effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.

Thaxtomin A (TA) is a cellulose biosynthesis inhibitor synthesized by the soil actinobacterium Streptomyces scabies, which is the main causal agent of potato common scab. TA is essential for the induction of scab lesions on potato tubers. When added to Arabidopsis thaliana cell cultures, TA induces an atypical programmed cell death (PCD). Although production of reactive oxygen species (ROS) often correlates with the induction of PCD, we observed a decrease in ROS levels following TA treatment. We show that this decrease in ROS accumulation in TA-treated cells is not due to the activation of antioxidant enzymes. Moreover, Arabidopsis cell cultures treated with hydrogen peroxide (H₂O₂) prior to TA treatment had significantly fewer dead cells than cultures treated with TA alone. This suggests that H₂O₂ induces biochemical or molecular changes in cell cultures that alleviate the activation of PCD by TA. Investigation of the cell wall mechanics using atomic force microscopy showed that H₂O₂ treatment can prevent the decrease in cell wall rigidity observed after TA exposure. While we cannot exclude the possibility that H₂O₂ may promote cell survival by altering the cellular redox environment or signaling pathways, our results suggest that H₂O₂ may inhibit cell death, at least partially, by reinforcing the cell wall to prevent or compensate for damages induced by TA.

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

Background: Thaxtomin A (TA), a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides) cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA. Results: Habituation to TA was performed by adding increasing levels of TA to cell cultures at the time of subculture over a period of 12 months. TA-habituated cells were then cultured in the absence of TA for more than three years. These cells displayed a reduced size and growth compared to control cells and had fragmented vacuoles filled with electron-dense material. Habituation to TA was associated with changes in the cell wall composition, with a reduction in cellulose and an increase in pectin levels. Remarkably, high level of resistance to TA was maintained in TA-habituated cells even after being cultured in the absence of TA. Moreover, these cells exhibited enhanced resistance to two other inhibitors of cellulose biosynthesis, dichlobenil and isoxaben. Analysis of gene expression in TA-habituated cells using an Affymetrix GeneChip Poplar Genome Array revealed that durable resistance to TA is associated with a major and complex reprogramming of gene expression implicating processes such as cell wall synthesis and modification, lignin and flavonoid synthesis, as well as DNA and chromatin modifications. Conclusions: We have shown that habituation to TA induced durable resistance to the bacterial toxin in poplar cells. TA-habituation also enhanced resistance to two other structurally different inhibitors of cellulose synthesis that were found to target different proteins. Enhanced resistance was associated with major changes in the expression of numerous genes, including some genes that are involved in DNA and chromatin modifications, suggesting that epigenetic changes might be involved in this process.