Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC 50 ) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.T he diseases Fusarium head blight (FHB) and root rot, caused by pathogenic ascomycete fungi of the genus Fusarium, such as Fusarium graminearum (Fg), are devastating diseases of cereal crops (1). Fusarium represents one of the most important cereal killers worldwide, exerting great economic and agronomic impact on global grain production and the grain industry. In addition to considerable yield losses, food quality is detrimentally affected by grain contamination with mycotoxins, which are produced by the fungi during plant infection (2-4). These contaminants represent a serious threat to human and animal health (5).Plant protection and toxin reduction strategies are presently mediated by chemical treatments, resistance breeding strategies, biological control, and genetic engineering. The latter relies on the use of antifungal transgenes, such as chitinase, defensins, polygalacturonase, and the use of mycotoxin detoxifying enzymes (6). However, the use of antifungal traits has not provided convincing practical solutions in terms of efficiency and reliability under agronomical practice.Currently, the application of systemic fungicides, such as sterol demethylation inhibitors (DMIs), is essential for controlling Fusarium diseases and thereby reaching the attainable production level of modern high-yield cultivars. DMI fungicides, such as tebuconazole, triadimefon, and prochloraz, act as ergosterol biosynthesis inhibitors because of cytochrome P450 lanosterol C-14α-demethylase (CYP51) binding, which subsequently disturbs fungal membrane integrity (7). Because of a shortage of alternative chemicals, D...

The Alphaproteobacterium Rhizobium radiobacter F4 (RrF4) was originally characterized as an endofungal bacterium in the beneficial endophytic Sebacinalean fungus Piriformospora indica. Although attempts to cure P. indica from RrF4 repeatedly failed, the bacterium can easily be grown in pure culture. Here, we report on RrF4's genome and the beneficial impact the free-living bacterium has on plants. In contrast to other endofungal bacteria, the genome size of RrF4 is not reduced. Instead, it shows a high degree of similarity to the plant pathogenic R. radiobacter (formerly: Agrobacterium tumefaciens) C58, except vibrant differences in both the tumor-inducing (pTi) and the accessor (pAt) plasmids, which can explain the loss of RrF4's pathogenicity. Similar to its fungal host, RrF4 colonizes plant roots without host preference and forms aggregates of attached cells and dense biofilms at the root surface of maturation zones. RrF4-colonized plants show increased biomass and enhanced resistance against bacterial leaf pathogens. Mutational analysis showed that, similar to P. indica, resistance mediated by RrF4 was dependent on the plant's jasmonate-based induced systemic resistance (ISR) pathway. Consistent with this, RrF4-and P. indica-induced pattern of defense gene expression were similar. In clear contrast to P. indica, but similar to plant growth-promoting rhizobacteria, RrF4 colonized not only the root outer cortex but also spread beyond the endodermis into the stele. On the basis of our findings, RrF4 is an efficient plant growth-promoting bacterium.

SummaryMicrorchidia (MORC) proteins comprise a family of proteins that have been identified in prokaryotes and eukaryotes. They are defined by two hallmark domains: a GHKL‐type ATPase and an S5‐fold. In plants, MORC proteins were first discovered in a genetic screen for Arabidopsis thaliana mutants compromised for resistance to a viral pathogen. Subsequent studies expanded their role in plant immunity and revealed their involvement in gene silencing and genome stabilization. Little is known about the role of MORC proteins of cereals, especially because knockout (KO) mutants were not available and assessment of loss of function relied only on RNAi strategies, which were arguable, given that MORC proteins in itself are influencing gene silencing. Here, we used a Streptococcus pyogenes Cas9 (SpCas9)‐mediated KO strategy to functionally study HvMORC1, one of the current seven MORC members of barley. Using a novel barley RNA Pol III‐dependent U3 small nuclear RNA (snRNA) promoter to drive expression of the synthetic single guide RNA (sgRNA), we achieved a very high mutation frequency in HvMORC1. High frequencies of mutations were detectable by target sequencing in the callus, the T0 generation (77%) and T1 generation (70%–100%), which constitutes an important improvement of the gene‐editing technology in cereals. Corroborating and extending earlier findings, SpCas9‐edited hvmorc1‐ KO barley, in clear contrast to Arabidopsis atmorc1 mutants, had a distinct phenotype of increased disease resistance to fungal pathogens, while morc1 mutants of either plant showed de‐repressed expression of transposable elements (TEs), substantiating that plant MORC proteins contribute to genome stabilization in monocotyledonous and dicotyledonous plants.

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 hvmor-c6a, 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.

Non-expressor of pathogenesis-related genes 1 (NPR1) is a key regulator of plant innate immunity and systemic disease resistance. The model for NPR1 function is based on experimental evidence obtained largely from dicots; however, this model does not fit all aspects of Poaceae family, which includes major crops such as wheat, rice and barley. In addition, there is little scientific data on NPR1's role in mutualistic symbioses. We assessed barley (Hordeum vulgare) HvNPR1 requirement during the establishment of mutualistic symbiosis between barley and beneficial Alphaproteobacterium Rhizobium radiobacter F4 (RrF4). Upon RrF4 root-inoculation, barley NPR1-knockdown (KD-hvnpr1) plants lost the typical spatiotemporal colonization pattern and supported less bacterial multiplication. Following RrF4 colonization, expression of salicylic acid marker genes were strongly enhanced in wild-type roots; whereas in comparison, KD-hvnpr1 roots exhibited little to no induction. Both basal and RrF4-induced root-initiated systemic resistance against virulent Blumeria graminis were impaired in leaves of KD-hvnpr1. Besides these immune-related differences, KD-hvnpr1 plants displayed higher root and shoot biomass than WT. However, RrF4-mediated growth promotion was largely compromised in KD-hvnpr1. Our results demonstrate a critical role for HvNPR1 in establishing a mutualistic symbiosis between a beneficial bacterium and a cereal crop.