Biology subjects

Krczal, G.

Publications and source records attributed to Krczal, G..

6 recordsLinked to original sources

bioRxiv2026

Functional RNA Interference in Hymenoscyphus fraxineus: Hairpin RNA-Induced Gene Silencing Of A Polyketide Synthase-like Gene

European ash dieback caused by the invasive ascomycete species Hymenoscyphus fraxineus poses the most prominent danger to common ash trees (Fraxinus excelsior) in Europe. The disease is widely distributed in Europe and currently no efficient management strategy is available. Host-induced gene silencing and exogenous dsRNA applications have shown great potential for controlling fungal diseases in crop plants. In this study, we reported in silico evidence for the presence of a functional RNA interference pathway in Hymenoscyphus fraxineus. Moreover, we showed that the transgenic expression of a double stranded RNA (dsRNA) leads to inhibition of translation of its target polyketide synthase-like gene, a fungal endogene. We explored whether the dsRNA could be introduced exogenously and demonstrated that H. fraxineus can take up externally applied dsRNA molecules. This study highlights the RNA interference mechanism in H. fraxineus and suggests exoRNA applications as a promising approach to control European ash dieback.

molecular biology↗

bioRxiv2025

Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

In planta gene expression via exogenous mRNAs has a wide range of potential biotechnological applications from genome editing to plant protection and to circumventing tissue culture. Yet, regarding mRNA delivery into intact plant cells, the purity, stability and translational efficiency of exogenous mRNAs appear as obstacles for effective exogenous mRNA application. Our study investigates the potential of exogenous mRNAs in mediating protein synthesis in plant cells. We applied unformulated, unmodified green fluorescent protein (GFP)-coding mRNA to monocot (Triticum aestivum, Hordeum vulgare, Zea mays) crops and dicot model plant Arabidopsis thaliana and observed GFP signals in all species, confirming the successful uptake and translation of mRNA into protein. This approach opens possibilities for broader, GM-free applications in plant science.

plant biology↗

bioRxiv2022

Beet curly top Iran virus Rep and V2 gene work as silencing suppressors through separate mechanisms

Beet curly top Iran Virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles mastreviruses, whereas the virion strand organization is close to curtoviruses. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) level. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains elusive in becurtoviruses. By screening all verified open reading frames in the BCTIV genome, we found that only V2 and Rep were able to suppress specific PTGS mechanisms, triggered by the expression of a partial or full-length sense-strand transcript of the target gene (S-PTGS). BCTIV-V2 could suppress S-PTGS more efficiently than BCTIV-Rep when then the target GFP gene is transiently expressed. On the other hand, S-PTGS is suppressed by Rep but not V2 when target GFP is only stably expressed. Deletional mutagenesis of BCTIV-Rep implicated that multiple domains are required for its VSR activity. Furthermore, neither V2 nor Rep could fully suppress local PTGS induced by inverted repeat targeting GFP (GFP-IR). Also, in a closer look at the spread of local silencing by GFP-IR, we observed that V2 or Rep are not able to suppress the movement of sRNAs. Nevertheless, Rep suppressed the systemic silencing induced by GFP-IR in 16C plants. Northern blot analyses showed that BCTIV-Rep inhibits silencing by mitigating sRNA production, whereas BCTIV-V2 does not alter sRNA levels. In summary, both the silencing phenotype and the molecular signatures of silencing implicate distinct modes of VSR activity of BCTIV-Rep and -V2.

plant biology↗

bioRxiv2022

Induction of promoter methylation and transcriptional gene silencing upon high pressure spraying of 24-nt small RNAs in Nicotiana benthamiana

Exogenous RNA application is a promising transgene-free approach for crop protection and improvement. In almost all applications reported so far, exogenous RNA molecules (double stranded RNAs or small RNAs) are applied to plants in order to trigger degradation of a given mRNA (of plant, pest or pathogen origin), in a process termed post-transcriptional gene silencing (PTGS). However, whether exogenous RNAs can also trigger epigenetic modifications to plants such as RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) remains largely unaddressed. Here, we provide evidence that high pressure spraying of a 24-nt short interfering RNA (siRNA) designed to target the CaMV 35S promoter that drives the expression of a GFP transgene, resulted in promoter RdDM in N. benthamiana. Moreover, the methylation at the target site spreads both up and downstream neighboring sites. Importantly, GFP expression was reduced in the sprayed leaf tissues. Small RNA sequencing excluded the presence siRNAs mapping to the GFP gene body and documented substantial amount of secondary RNAs in the promoter, suggesting that the GFP silencing was transcriptional rather than post-transcriptional. Our study provides a proof of principle for spray induced epigenetic modification (SPIEM) that could be used in modern crop breeding platforms.

plant biology↗

bioRxiv2021

High-pressure sprayed siRNA triggers influence the efficiency but not the profile of transitive silencing

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3 of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5 and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3 end of the target RNA. SIGNIFICANCE STATEMENTThis work answers a long-standing question about the role of siRNA triggers in initiating transitive silencing. By using high-pressure spraying-mediated delivery of synthetic siRNAs, we provided experimental evidence that target position of 22-nt-long primary siRNAs influences the efficiency of RDR6-driven double stranded RNA (dsRNA) synthesis, but it does not change the profile of accumulating secondary siRNAs originating from processing of RDR6-produced dsRNA.

plant biology↗

bioRxiv2020

The anticipated potential nuclear localization sequence of 'Candidatus Phytoplasma mali SAP11-like protein is required for TCP binding but not for transport into the nucleus

AO_SCPLOWBSTRACTC_SCPLOWThe plant pathogen Candidatus Phytoplasma mali ( Ca. P. mali) is the causing agent of apple proliferation that leads to heavy damage in apple production all over Europe. To identify and analyze effector proteins of plant pathogens is an important strategy in plant disease research. Here, we report that the SAP11-like protein of Ca. P. mali induces crinkled leaves and siliques and witches broom symptoms in transgenic Arabidopsis thaliana (A. thaliana) plants and binds to 6 members of class I and all members of class II TCP (TEOSINE BRANCHES/ CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of A. thaliana in yeast two-hybrid assays. Moreover, we demonstrate that the protein localizes actively into the plant nucleus without requiring the nuclear leader sequence (NLS). We also identified a 17 amino acid stretch previously predicted to be a nuclear leader sequence that is important for the binding of some of the TCPs and also responsible for the crinkled leaf and silique phenotype in transgenic A. thaliana.

plant biology↗