Development of transgenic Paulownia trees expressing antimicrobial thionin genes for enhanced resistance to fungal infections using chitosan nanoparticles.

There is an increasing focus on genetically altering Paulownia trees to enhance their resistance against fungal infections, given their rapid growth and quality wood production. The aim of this research was to establish a technique for incorporating two antimicrobial thionin genes, namely thionin-60 (thio-60) and thionin-63 (thio-63), into Paulownia tomentosa and Paulownia hybrid 9501 through the utilization of chitosan nanoparticles. The outcomes revealed the successful gene transfer into Paulownia trees utilizing chitosan nanoparticles. The effectiveness of thionin proteins against plant pathogens Fusarium and Aspergillus was examined, with a specific focus on Fusarium equiseti due to limited available data. In non-transgenic Paulownia species, the leaf weight inhibition percentage varied from 25 to 36 %, whereas in transgenic species, it ranged from 22 to 7 %. In general, Paulownia species expressing thio-60 displayed increased resistance to F. equiseti, while those expressing thio-63 exhibited heightened resistance to A. niger infection. The thionin proteins displayed a strong affinity for the phospholipid bilayer of the fungal cell membrane, demonstrating their capability to disrupt its structure. The transgenic plants created through this technique showed increased resistance to fungal infections. Thionin-60 demonstrated superior antifungal properties in comparison to thio-63, being more effective at disturbing the fungal cell membrane. These findings indicate that thio-60 holds potential as a novel antifungal agent and presents a promising approach for enhancing the antimicrobial traits of genetically modified Paulownia trees.

Arabidopsis thionin-like genes are involved in resistance against the beet-cyst nematode (Heterodera schachtii).

Plants express various antimicrobial peptides including thionins to protect themselves against pathogens. It was recently found that, in addition to four thionin genes, Arabidopsis contains 67 thionin-like (ThiL) genes including six pseudogenes. It is known that thionins have antimicrobial activity and are part of the plant defense system, however, nothing is known about ThiL genes. In this study, we present a bioinformatic analysis of the (ThiL) gene family in Arabidopsis. We identified 15 different motifs which positioned the ThiL peptides in four groups. A comparison of amino acid sequences showed that the ThiL peptides are actually more similar to the acidic domain of thionin proproteins than to the thionin domain. We selected 10 ThiL genes to study the expression and possible function in the Arabidopsis plant. RT-PCR and promoter:GUS fusions showed that most genes were expressed at a very low level but in several organs and at different developmental stages. Some genes were also expressed in syncytia induced by the beet cyst nematode Heterodera schachti in roots while others were downregulated in syncytia. Some overexpression lines supported lower number of nematodes that developed on the roots after inoculation. Two of the genes resulted in a strong hypersensitive response when infiltrated into leaves of Nicotiana benthamiana. These results indicate that ThiL genes might be involved in the response to biotic stress. ThiL genes have been expanded in the Brassicales and specifically the Brassicaceae. The most extreme example is the CRP2460 subfamily that contains 28 very closely related genes from Arabidopsis which are mostly the result of tandem duplications.

A gamma-thionin protein from apple, MdD1, is required for defence against S-RNase-induced inhibition of pollen tube prior to self/non-self recognition.

Apple exhibits S-RNase-mediated self-incompatibility. Although the cytotoxic effect of S-RNase inside the self-pollen tube has been studied extensively, the underlying defence mechanism in pollen tube in Rosaceae remains unclear. On exposure to stylar S-RNase, plant defence responses are activated in the pollen tube; however, how these are regulated is currently poorly understood. Here, we show that entry of both self and non-self S-RNase into pollen tubes of apple (Malus domestica) stimulates jasmonic acid (JA) production, in turn inducing the accumulation of MdMYC2 transcripts, a transcription factor in the JA signalling pathway widely considered to be involved in plant defence processes. MdMYC2 acts as a positive regulator in the pollen tube activating expression of MdD1, a gene encoding a defence protein. Importantly, MdD1 was shown to bind to the RNase activity sites of S-RNase leading to inhibition of enzymatic activity. This work provides intriguing insights into an ancient defence mechanism present in apple pollen tubes where MdD1 likely acts as a primary line of defence to inhibit S-RNase cytotoxicity prior to self/non-self recognition.

In Silico Analyses of Rice Thionin Genes and the Antimicrobial Activity of OsTHION15 Against Phytopathogens.

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). RESULTS: Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. CONCLUSIONS: Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.

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.