Barbara De Coninck

Plant pathogenesis-related (PR) proteins: A focus on PR peptides

The novel classes of plant pathogenesis-related (PR) proteins identified during the last decade also include novel peptide families. This review specifically focuses on these pathogenesis-related peptides, including proteinase inhibitors (PR-6 family), plant defensins (PR-12 family), thionins (PR-13 family) and lipid transfer proteins (PR-14 family). For each family of PR peptides, the general features concerning occurrence, expression and possible functions of their members are described. Next, more specifically the occurrence of each PR peptide family in the model plant Arabidopsis thaliana is discussed. Single-gene studies performed on particular gene members of a PR peptide family are reported. In addition, expression data of yet undescribed gene members of that particular PR peptide family are presented by consultation of publicly available micro-array databases. Finally an update is provided on the potential role of these PR peptides in A. thaliana, with a focus on their possible involvement in plant defense.

Unraveling the Difference between Invertases and Fructan Exohydrolases: A Single Amino Acid (Asp-239) Substitution Transforms Arabidopsis Cell Wall Invertase1 into a Fructan 1-Exohydrolase

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions

A unifying peptide classification system is proposed, reflecting the huge diversity of plant peptides. Peptides fulfill a plethora of functions in plant growth, development, and stress responses. They act as key components of cell-to-cell communication, interfere with signaling and response pathways, or display antimicrobial activity. Strikingly, both the diversity and amount of plant peptides have been largely underestimated. Most characterized plant peptides to date acting as small signaling peptides or antimicrobial peptides are derived from nonfunctional precursor proteins. However, evidence is emerging on peptides derived from a functional protein, directly translated from small open reading frames (without the involvement of a precursor) or even encoded by primary transcripts of microRNAs. These novel types of peptides further add to the complexity of the plant peptidome, even though their number is still limited and functional characterization as well as translational evidence are often controversial. Here, we provide a comprehensive overview of the reported types of plant peptides, including their described functional and structural properties. We propose a novel, unifying peptide classification system to emphasize the enormous diversity in peptide synthesis and consequent complexity of the still expanding knowledge on the plant peptidome.

Genome-Wide Characterization of ISR Induced in Arabidopsis thaliana by Trichoderma hamatum T382 Against Botrytis cinerea Infection

In this study, the molecular basis of the induced systemic resistance (ISR) in Arabidopsis thaliana by the biocontrol fungus Trichoderma hamatum T382 against the phytopathogen Botrytis cinerea B05-10 was unraveled by microarray analysis both before (ISR-prime) and after (ISR-boost) additional pathogen inoculation. The observed high numbers of differentially expressed genes allowed us to classify them according to the biological pathways in which they are involved. By focusing on pathways instead of genes, a holistic picture of the mechanisms underlying ISR emerged. In general, a close resemblance is observed between ISR-prime and systemic acquired resistance, the systemic defense response that is triggered in plants upon pathogen infection leading to increased resistance toward secondary infections. Treatment with T. hamatum T382 primes the plant (ISR-prime), resulting in an accelerated activation of the defense response against B. cinerea during ISR-boost and a subsequent moderation of the B. cinerea induced defense response. Microarray results were validated for representative genes by qRT-PCR. The involvement of various defense-related pathways was confirmed by phenotypic analysis of mutants affected in these pathways, thereby proving the validity of our approach. Combined with additional anthocyanin analysis data these results all point to the involvement of the phenylpropanoid pathway in T. hamatum T382-induced ISR.

What lies beneath: belowground defense strategies in plants

Diseases caused by soil-borne pathogens result worldwide in significant yield losses in economically important crops. In contrast to foliar diseases, relatively little is known about the nature of root defenses against these pathogens. This review summarizes the current knowledge on root infection strategies, root-specific preformed barriers, pathogen recognition, and defense signaling. Studies reviewed here suggest that many commonalities as well as differences exist in defense strategies employed by roots and foliar tissues during pathogen attack. Importantly, in addition to pathogens, plant roots interact with a plethora of non-pathogenic and symbiotic microorganisms. Therefore, a good understanding of how plant roots interact with the microbiome would be particularly important to engineer resistance to root pathogens without negatively altering root-beneficial microbe interactions.

RNAseq‐based transcriptome analysis of Lactuca sativa infected by the fungal necrotroph Botrytis cinerea

The fungal pathogen Botrytis cinerea establishes a necrotrophic interaction with its host plants, including lettuce (Lactuca sativa), causing it to wilt, collapse and eventually dry up and die, which results in serious economic losses. Global expression profiling using RNAseq and the newly sequenced lettuce genome identified a complex network of genes involved in the lettuce-B. cinerea interaction. The observed high number of differentially expressed genes allowed us to classify them according to the biological pathways in which they are implicated, generating a holistic picture. Most pronounced were the induction of the phenylpropanoid pathway and terpenoid biosynthesis, whereas photosynthesis was globally down-regulated at 48 h post-inoculation. Large-scale comparison with data available on the interaction of B. cinerea with the model plant Arabidopsis thaliana revealed both general and species-specific responses to infection with this pathogen. Surprisingly, expression analysis of selected genes could not detect significant systemic transcriptional alterations in lettuce leaves distant from the inoculation site. Additionally, we assessed the response of these lettuce genes to a biotrophic pathogen, Bremia lactucae, revealing that similar pathways are induced during compatible interactions of lettuce with necrotrophic and biotrophic pathogens.

Arabidopsis thaliana plant defensin AtPDF1.1 is involved in the plant response to biotic stress.

*Previously, it was shown that the Arabidopsis thaliana plant defensins AtPDF1.1 (At1g75830) and AtPDF1.2a (At5g44420) exert in vitro antimicrobial properties and that their corresponding genes are expressed in seeds and induced in leaves upon pathogen attack, respectively. *In this study, the expression profile of both AtPDF1.1 and AtPDF1.2a is analysed in wild-type plants upon different stress-related treatments and the effect of modulation of their expression in transgenic plants is examined in both host and nonhost resistance. *AtPDF1.1, which was originally considered to be seed-specific, is demonstrated to be locally induced in leaves upon fungal attack and exhibits an expression profile distinct from that of AtPDF1.2a, a gene frequently used as marker for the ethylene/jasmonate-mediated signaling pathway. Transgenic plants with modulated AtPDF1.1 or AtPDF1.2a gene expression show no altered phenotype upon Botrytis cinerea inoculation. However, constitutive overexpression of AtPDF1.1 in A. thaliana leads to a reduction in symptoms caused by the nonhost Cercospora beticola causing non-spreading spots on A. thaliana leaves. *These results indicate that AtPDF1.1 and AtPDF1.2a clearly differ regarding their expression profile and functionality in planta. It emphasizes the additional level of complexity and fine-tuning within the highly redundant plant defensin genes in A. thaliana.

The Arabidopsis thaliana RNA Editing Factor SLO2, which Affects the Mitochondrial Electron Transport Chain, Participates in Multiple Stress and Hormone Responses

Recently, we reported that the novel mitochondrial RNA editing factor SLO2 is essential for mitochondrial electron transport, and vital for plant growth through regulation of carbon and energy metabolism. Here, we show that mutation in SLO2 causes hypersensitivity to ABA and insensitivity to ethylene, suggesting a link with stress responses. Indeed, slo2 mutants are hypersensitive to salt and osmotic stress during the germination stage, while adult plants show increased drought and salt tolerance. Moreover, slo2 mutants are more susceptible to Botrytis cinerea infection. An increased expression of nuclear-encoded stress-responsive genes, as well as mitochondrial-encoded NAD genes of complex I and genes of the alternative respiratory pathway, was observed in slo2 mutants, further enhanced by ABA treatment. In addition, H2O2 accumulation and altered amino acid levels were recorded in slo2 mutants. We conclude that SLO2 is required for plant sensitivity to ABA, ethylene, biotic, and abiotic stress. Although two stress-related RNA editing factors were reported very recently, this study demonstrates a unique role of SLO2, and further supports a link between mitochondrial RNA editing events and stress response.

Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance

Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.

Synergistic Activity of the Plant Defensin HsAFP1 and Caspofungin against Candida albicans Biofilms and Planktonic Cultures.

Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.