Biology subjects

Kogel, K.-H.

Publications and source records attributed to Kogel, K.-H..

11 recordsLinked to original sources

bioRxiv2024

Broad-scale phenotyping in Arabidopsis reveals varied involvement of RNA interference across diverse plant-microbe interactions

RNA interference (RNAi) is a crucial mechanism that can contribute to immunity against infectious microbes through the action of DICER-LIKE (DCL) and ARGONAUTE (AGO) proteins. In the case of the fungal pathogen Botrytis cinerea and the oomycete Hyaloperonospora arabidopsidis, plant DCL and AGO proteins have proven roles as negative regulators of immunity, suggesting functional specialization of these proteins. To address this aspect in a broader taxonomic context, we characterized the colonization pattern of an informative set of DCL and AGO loss-of-function mutants in Arabidopsis thaliana upon infection with a panel of pathogenic microbes with different lifestyles, and a fungal mutualist. Our results revealed that AGO1 and AGO4 function as positive regulators of immunity to a bacterial and a fungal pathogen, respectively. Additionally, AGO2 and AGO10 positively modulated the colonization by a fungal mutualist. Therefore, analysing the role of RNAi across a broader range of plant-microbe interactions has identified previously unknown functions for AGO proteins. For some pathogen interactions, however, all tested mutants exhibited wild type-like infection phenotypes, suggesting that the roles of AGO and DCL proteins in these interactions may be more complex to elucidate.

plant biology↗

bioRxiv2023

A robust pipeline for the systematic validation of sRNA effectors suggests cross-kingdom communication in the symbiosis of Serendipita indica with Arabidopsis thaliana

Bidirectional communication between pathogenic microbes and their plant hosts via small (s)RNA-mediated cross-kingdom RNA interference (ckRNAi) is one key element for successful host colonisation. However, whether mutualistic fungi from the Serendipitaceae family, known for their extremely broad host range, employ sRNAs to colonize plant roots is still under discussion. To address this question, we developed a pipeline to validate the accumulation, translocation, and activity of fungal sRNAs in post-transcriptional silencing of Arabidopsis thaliana genes. Using stem-loop PCR, we detected the expression of a specific set of Serendipita indica (Si)sRNAs, targeting host genes involved in cell wall organization, hormonal signalling regulation, immunity, and gene regulation. To confirm the gene silencing activity of these sRNA in plant cells, SisRNAs were transiently expressed in protoplasts. Stem-loop PCR proved the expression of sRNAs, while qPCR validated post-transcriptional gene silencing of their predicted target genes. Furthermore, ARGONAUTE 1 immunoprecipitation (AtAGO1-IP) revealed the loading of fungal SisRNAs into the plant RNAi machinery, suggesting the translocation of SisRNA from the fungus into root cells. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors in plant-microbe interactions and further suggests cross-kingdom communication in the Sebacinoid symbiosis. HighlightSmall RNAs of the beneficial fungus Serendipita indica are translocated and silence Arabidopsis genes at the onset of the interaction, revealing cross-kingdom communication in sebacinoid symbiosis.

plant biology↗

bioRxiv2023

Designer antisense circRNAGFP reduces GFP protein abundance in transgenic Arabidopsis protoplasts in a sequence-specific manner, independent of RNAi pathways

Circular RNAs (circRNAs) are single-stranded RNA molecules characterised by their covalently closed structure and are emerging as key regulators of cellular processes in mammals, including gene expression, protein function and immune responses. Recent evidence suggests that circRNAs also play significant roles in plants, influencing development, nutrition, biotic stress resistance, and abiotic stress tolerance. However, the potential of circRNAs to modulate target protein abundance in plants remains largely unexplored. In this study, we investigated the potential of designer circRNAs to modulate target protein abundance in plants using Arabidopsis as a model system. We demonstrate that treatment with a 50 nt circRNAGFP, containing a 30 nt GFP antisense sequence stretch, results in reduced GFP reporter target protein abundance in a dose- and sequence-dependent manner. Notably, a single-stranded open isoform of circRNAGFP had little effect on protein abundance, indicating the importance of the closed circular structure. Additionally, circRNAGFP also reduced GFP abundance in Arabidopsis mutants defective in RNA interference (RNAi), suggesting that circRNA activity is independent of the RNAi pathway. We also show that circRNA, unlike dsRNA, does not induce pattern-triggered immunity (PTI) in plants. Findings of this proof-of-principle study together are crucial first steps in understanding the potential of circRNAs as versatile tools for modulating gene expression and offer exciting prospects for their application in agronomy, particularly for enhancing crop traits through metabolic pathway manipulation. HighlightsWe demonstrate the potential of non-immunogenic circRNA as a tool for targeted gene regulation in plants, where circRNA acts in an isoform- and sequence-specific manner, paving the way for future agronomic applications.

plant biology↗

bioRxiv2022

A key regulator with a MIF-like domain orchestrates cellular differentiation and virulence in the fungal pathogen Magnaporthe oryzae

MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) is a pleiotropic protein with chemotactic, pro-inflammatory, and growth-promoting activities first discovered in mammals. In parasites, MIF homologs are involved in immune evasion and pathogenesis. Here, we present the first comprehensive analysis of a MIF protein from the devastating plant pathogen Magnaporthe oryzae (Mo). The fungal genome encodes a single MIF protein (MoMIF1) that, unlike the human homolog, harbors multiple low-complexity regions (LCRs) and is unique to Ascomycota. Following infection, MoMIF1 is expressed in the biotrophic phase of the fungus, and is strongly down-regulated during subsequent necrotrophic growth in leaves and roots. We show that MoMIF1 is secreted during plant infection, affects the production of the mycotoxin tenuazonic acid and inhibits plant cell death. Our results show that MoMIF1 is a novel key regulator of fungal virulence that maintains the balance between biotrophy and necrotrophy during the different phases of fungal infection.

plant biology↗

bioRxiv2022

Analysis of codon usage and allele frequencies reveal the double-edged nature of cross-kingdom RNAi

BackgroundIn recent years, a new class of small 21- to 24-nt-(s)RNAs has been discovered from microbial pathogens that interfere with their hosts gene expression during infection, reducing the hosts defence in a process called cross-kingdom RNA interference (ckRNAi). According to this model, microbial sRNAs should exert selection pressure on plants so that gene sequences that reduce complementarity to sRNAs are preferred. In this paper, we test this consequence of the ckRNA model by analyzing changes to target sequences considering codon usage and allele frequencies in the model system Arabidopsis thaliana (At) - Hyaloperonospora arabidopsidis (Ha) and Hordeum vulgare (Hv) - Fusarium graminearum (Fg). In both pathosystems, some selected sRNA and their corresponding target have been described and experimentally validated, while the lengthy methodology prevents the analysis of all discovered sRNAs. To expand the understanding of ckRNAi, we apply a new in silico approach that integrates the majority of sRNAs. ResultsWe calculated the probability (PCHS) that synonymous host plant codons in a predicted sRNA target region would show the same or stronger complementarity as actually observed and compared this probability to sets of virtual analogous sRNAs. For the sets of Ha and Fg sRNAs, there was a significant difference in codon usage in their plant gene target regions (for Ha: PCHS 24.9% lower than in the virtual sets; for Fg: PCHS 19.3% lower than in the virtual sets), but unexpectedly for both sets of microbial sRNA we found a tendency towards codons with an unexpectedly high complementarity. To distinguish between complementarity caused by balancing sRNA-gene coevolution and directional selection we estimated Wrights F-statistic (FST), a measurement of population structure, in which positive deviations from the background indicate directional and negative deviations balancing selection at the respective loci. We found a negative correlation between PCHS and FST (p=0.03) in the At-Ha system indicating deviations from codon usage favoring complementarity are generally directionally selected. ConclusionThe directional selection of complementary codons in host plants suggests an evolutionary pressure to facilitate silencing by exogenous microbial sRNAs, which is not consistent with the anticipated biological role of pathogen sRNAs as exclusively effectors in cross-kingdom RNAi. To resolve this conflict, we propose an extended model in which microbial sRNAs are perceived by plants via RNA interference and, via coevolution, primarily help to fine-tune plant gene expression.

plant biology↗

bioRxiv2022

Identification of novel leaf rust seedling resistance loci in Iranian bread wheat germplasm using genome-wide association mapping

Leaf or brown rust caused by Puccinia triticina Eriks. (Pt) is a major biotic constraint threatening bread wheat production worldwide. The continued evolution of new races of Pt necessitates a constant search for the identification of new resistance genes, or QTLs, to enhance the resistance durability of bread varieties. On a panel of 320 bread wheat accessions, we used a genome-wide association study (GWAS) technique to map loci associated with Pt resistance using single-nucleotide polymorphism markers (SNPs) generated by genotyping-by-sequencing (GBS). The panel was tested with five Pt races gathered from different regions of IRAN to identify loci associated with seedling resistance. After estimating genetic relatedness and population structure among accessions, GWAS discovered a total of 19 SNPs on chromosomes 1B, 2B, 3A, 3B, 4A, 5B, 5D, 6A, 6B, 6D, 7B, and 7D that were significantly associated with seedling stage resistance. The three SNP markers rs12954, rs34220, and rs42447 on chromosomes 5D, 6A, and 7D, respectively, associated with resistance to Pt race PKTTS expressing potential new loci for leaf rust resistance. Overall, this research gives an integrated perspective of leaf rust resistance resources in Iranian bread wheat and recognizes new resistance loci that will be valuable to expand the set of resistance genes available to control this serious disease.

plant biology↗

bioRxiv2021

Fusarium graminearum DICER-like-dependent sRNAs are required for the suppression of host immune genes and full virulence

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing. Significance StatementFusarium graminearum is one of the most devastating fungal pathogens in cereals, while understanding the mechanisms of fungal pathogenesis is a prerequisite for developing efficient and environmentally friendly crop protection strategies. We show exploratory data suggesting that fungal small RNAs play a critical role in Fusarium virulence by suppressing plant immunity.

microbiology↗

bioRxiv2021

Evaluation of dsRNA delivery methods for targeting macrophage migration inhibitory factor MIF in RNAi-based aphid control

Macrophage migration inhibitory factors (MIF) are multifunctional proteins regulating major processes in mammals, including activation of innate immune responses. In invertebrates, MIF proteins participate in the modulation of host immune responses when secreted by parasitic organisms, such as aphids. In this study, we assessed the possibility to use MIF genes as targets for RNA interference (RNAi)-based control of the grain aphid Sitobion avenae (Sa) on barley (Hordeum vulgare). When nymphs were fed on artificial diet containing double-stranded (ds)RNAs (SaMIF-dsRNAs) that target sequences of the three MIF genes SaMIF1, SaMIF2 and SaMIF3, they showed higher mortality rates and these rates correlated with reduced MIF transcript levels as compared to the aphids feeding on artificial diet containing a control dsRNA (GFP-dsRNA). Comparison of different feeding strategies showed that nymphs survival was not altered when they fed from barley seedlings sprayed with SaMIF-dsRNAs, suggesting they did not effectively take up dsRNA from the sieve tubes of these plants. Furthermore, aphids survival was also not affected when the nymphs fed on leaves supplied with dsRNA via basal cut ends of barley leaves. Consistent with this finding, the use of sieve-tube-specific YFP-labeled Arabidopsis reporter lines confirmed that fluorescent 21 nt dsRNACy3 supplied via petioles co-localized with xylem structures, but not with phloem tissue. Our results suggest that MIF genes are a potential target for insect control and also imply that application of naked dsRNA to plants for aphid control is inefficient. More efforts should be put into the development of effective dsRNA formulations.

plant biology↗

bioRxiv2020

Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

Small (s)RNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through expression in transgenic plants and targeted gene silencing (host-induced gene silencing, HIGS). While HIGS strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unknown. Here we show that extracellular vesicles (EVs) purified from Arabidopsis thaliana plants expressing CYP3RNA, a dsRNA originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. These transgene specific siRNAs had a length of 21 and 22 nucleotides with a bias towards 5-uracil (U) and 5-adenine (A). Notably, stringent protease and RNase treated EV fractions contained >70% less CYP3RNA-derived siRNAs, suggesting the presence of co-purified extravesicular nucleoprotein complexes stabilizing siRNAs outside of EVs. In addition, mutants of the ESCRT-III complex showed a loss of HIGS-mediated disease resistance and EVs isolated from these mutants were free of CYP3RNA-derived siRNAs. Together, these findings support the view that endosomal vesicle trafficking is required for HIGS mediating the transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells probably in an EV-independent manner.

plant biology↗

bioRxiv2019

Various components of the RNAi pathway are required for conidiation, ascosporogenesis, virulence, DON production and SIGS-mediated fungal inhibition by exogenous dsRNA in the Head Blight pathogen Fusarium graminearum

Gene silencing through RNA interference (RNAi) shapes many biological processes in filamentous fungi, including pathogenicity. In this study we explored the requirement of key components of fungal RNAi machinery, including DICER-like 1 and 2 (FgDCL1, FgDCL2), ARGONAUTE 1 and 2 (FgAGO1, FgAGO2), AGO-interacting protein FgQIP (QDE2-interacting protein), RecQ helicase (FgQDE3), and four RNA-dependent RNA polymerases (FgRdRP1, FgRdRP2, FgRdRP3, FgRdRP4), in the ascomycete mycotoxin-producing fungal pathogen Fusarium graminearum (Fg) for sexual and asexual multiplication, pathogenicity, and its sensitivity to double-stranded (ds)RNA. We corroborate and extend earlier findings that conidiation, ascosporogenesis and Fusarium Head Blight (FHB) symptom development require an operable RNAi machinery. The involvement of RNAi in conidiation is dependent on environmental conditions as it is detectable only under low light (< 2 {micro}mol m-2s-1). Although both DCLs and AGOs partially share their functions, the sexual ascosporogenesis is mediated primarily by FgDCL1 and FgAGO2, while FgDCL2 and FgAGO1 contribute to asexual conidia formation and germination. FgDCL1 and FgAGO2 also account for pathogenesis as their knock-out (KO) results in reduced FHB development. Apart from KO mutants{Delta} dcl2 and{Delta} ago1, mutants{Delta} rdrp2, {Delta}rdrp3, {Delta}rdrp4, {Delta}qde3 and{Delta} qip are strongly compromised for conidiation, while KO mutations in all RdPRs, QDE3 and QIP strongly affect ascosporogenesis. Analysis of trichothecenes mycotoxins in wheat kernels showed that the relative amount of deoxynivalenol (DON), calculated as [DON] per amount of fungal genomic DNA, was reduced in all spikes infected with RNAi mutants, suggesting the possibility that the fungal RNAi pathways affect Fgs DON production in wheat spikes. Moreover, gene silencing by exogenous target gene specific dsRNA (spray-induced gene silencing, SIGS) is dependent on fungal DCLs, AGOs, and QIP, but not on QDE3. Together these data show that in F. graminearum different key components of the RNAi machinery are crucial in different steps of fungal development and pathogenicity.

pathology↗

bioRxiv2019

Discovery of interaction-related sRNAs and their targets in the Brachypodium distachyon and Magnaporthe oryzae pathosystem

Microbial pathogens secrete small RNA (sRNA) effectors into plant hosts to aid infection by silencing transcripts of immunity and signaling-related genes through RNA interference (RNAi). Similarly, sRNAs from plant hosts have been shown to contribute to plant defense against microbial pathogens by targeting transcripts involved in virulence. This phenomenon is called bidirectional RNA communication or cross kingdom RNAi (ckRNAi). How far this RNAi-mediated mechanism is evolutionarily conserved is the subject of controversial discussions. We examined the bidirectional accumulation of sRNAs in the interaction of the hemibiotrophic rice blast fungus Magnaporthe oryzae (Mo) with the grass model plant Brachypodium distachyon (Bd). By comparative deep sequencing of sRNAs and mRNAs from axenic fungal cultures and infected leaves and roots, we found a wide range of fungal sRNAs that accumulated exclusively in infected tissues. Amongst those, 20-21 nt candidate sRNA effectors were predicted in silico by selecting those Mo reads that had complementary mRNA targets in Bd. Many of those mRNAs predicted to be targeted by Mo sRNAs were differentially expressed, particularly in the necrotrophic infection phase, including gene transcripts involved in plant defense responses and signaling. Vice versa, by applying the same strategy to identify Bd sRNA effectors, we found that Bd produced sRNAs targeting a variety of fungal transcripts, encoding fungal cell wall components, virulence genes and transcription factors. Consistent with function as effectors of these Bd sRNAs, their predicted fungal targets were significantly down-regulated in the infected tissues compared to axenic cultures, and deletion mutants for some of these target genes showed heavily impaired virulence phenotypes. Overall, this study provides the first experimentally-based evidence for bidirectional ckRNAi in a grass-fungal pathosystem, paving the way for further validation of identified sRNA-target duplexes and contributing to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture on staple crops.\n\nAuthor SummaryIn the present work, we provide first experimental evidence for bidirectional RNA communication in a grass-fungal pathosystem. We deployed the monocotyledonous plant Brachypodium distachyon, which is a genetic model for the staple crops wheat and rice, to investigate the interaction-related sRNAs for their role in RNA communication. By applying a previously published bioinformatics pipeline for the detection of sRNA effectors we identified potential plant targets for fungal sRNAs and vice versa, fungal targets for plant sRNAs. Inspection of the respective targets confirmed their downregulation in infected relative to uninfected tissues and fungal axenic cultures, respectively. By focusing on potential fungal targets, we identified several genes encoding fungal cell wall components, virulence proteins and transcription factors. The deletion of those fungal targets has already been shown to produce disordered virulence phenotypes. Our findings establish the basis for further validation of identified sRNA-mRNA target duplexes and contribute to the emerging research on naturally occurring cross-kingdom communication and its implications for agriculture.

plant biology↗