Browsing by Person "Koch, Aline"

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CRISPR/SpCas9‐mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity, transcriptional gene silencing and plant growth
(2021) Galli, Matteo; Martiny, Engie; Imani, Jafargholi; Kumar, Neelendra; Koch, Aline; Steinbrenner, Jens; Kogel, Karl‐Heinz
The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9‐mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium‐mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9‐induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo‐/heteromers with other HvMORCs and interact with components of the RNA‐directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.
Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum
(2022) Schlemmer, Timo; Lischka, Richard; Wegner, Linus; Ehlers, Katrin; Biedenkopf, Dagmar; Koch, Aline; Schlemmer, Timo; Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany; Lischka, Richard; Centre for BioSystems, Land Use and Nutrition, Institute of Phytopathology, Justus Liebig University, Giessen, Germany; Wegner, Linus; Intitute of Botany, Justus Liebig University, Giessen, Germany; Ehlers, Katrin; Intitute of Botany, Justus Liebig University, Giessen, Germany; Biedenkopf, Dagmar; Centre for BioSystems, Land Use and Nutrition, Institute of Phytopathology, Justus Liebig University, Giessen, Germany; Koch, Aline; Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany
Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley ( Hordeum vulgare ) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum . Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.
Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles to assess their role in RNA spray-based crop protection
(2021) Schlemmer, Timo; Barth, Patrick; Weipert, Lisa; Preußer, Christian; Hardt, Martin; Möbus, Anna; Busche, Tobias; Koch, Aline
The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.