Summary
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/
Sp
Cas9‐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/
Sp
Cas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation,
Sp
Cas9‐induced mutation in both
HvMORC1
and
HvMORC6a
genes was observed in 58% of T0 plants. Subsequent infection assays showed that
Hv
MORC6a 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
Hv
MORC1 and
Hv
MORC6a form distinct nucleocytoplasmic homo‐/heteromers with other
Hv
MORCs 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.
RNA-based disease control has shown great potential for controlling pest and diseases in crop plants. While delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a ...promising concept, it requires the generation of transgenic crop plants, which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Focusing on agronomic important barley -
Fusarium
spec. pathosystems, we have sought for alternative strategies to apply dsRNAs for fungal control. Recently, we have demonstrated that a spray application of a long noncoding dsRNA termed CYP3RNA, which targets the three fungal
Cytochrome P450 lanosterol C-14
α
-demethylase
genes
FgCYP51A
,
FgCYP51B
, and
FgCYP51C
, inhibits
Fusarium graminearum
(
Fg
) on barley leaves (Koch et al.,
PLoS Pathogens, 12
, e1005901,
2016
). Here we show that another Fusarium species,
F. culmorum
(
Fc
), also is sensitive to
CYP51
-derived dsRNAs. Treating
Fc
with various dsRNAs targeting the genes
FcCYP51A
,
FcCYP51B
and
FcCYP51C
was destructive to the fungus and resulted in growth retardation in
in vitro
cultures. We discuss important consequences of this finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential for novel plant protection strategies.
Summary
RNA
interference (
RNA
i) has emerged as a powerful genetic tool for scientific research over the past several years. It has been utilized not only in fundamental research for the assessment ...of gene function, but also in various fields of applied research, such as human and veterinary medicine and agriculture. In plants,
RNA
i strategies have the potential to allow manipulation of various aspects of food quality and nutritional content. In addition, the demonstration that agricultural pests, such as insects and nematodes, can be killed by exogenously supplied
RNA
i targeting their essential genes has raised the possibility that plant predation can be controlled by lethal
RNA
i signals generated
in planta
. Indeed, recent evidence argues that this strategy, called host‐induced gene silencing (
HIGS
), is effective against sucking insects and nematodes; it also has been shown to compromise the growth and development of pathogenic fungi, as well as bacteria and viruses, on their plant hosts. Here, we review recent studies that reveal the enormous potential
RNA
i strategies hold not only for improving the nutritive value and safety of the food supply, but also for providing an environmentally friendly mechanism for plant protection.
We report the identification, cloning, heterologous expression and functional characterization of a novel antifungal peptide named lucimycin from the common green bottle fly
. The lucimycin cDNA was ...isolated from a library of genes induced during the innate immune response in
larvae, which are used as therapeutic maggots. The peptide comprises 77 amino acid residues with a molecular mass of 8.2 kDa and a p
of 6.6. It is predicted to contain a zinc-binding motif and to form a random coil, lacking β-sheets or other secondary structures. Lucimycin was active against fungi from the phyla Ascomycota, Basidiomycota and Zygomycota, in addition to the oomycete
, but it was inactive against bacteria. A mutant version of lucimycin, lacking the four C-terminal amino acid residues, displayed 40-fold lower activity. The activity of lucimycin against a number of highly-destructive plant pathogens could be exploited to produce transgenic crops that are resistant against fungal diseases.
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.
Recognition of specific molecule signatures of microbes, including pathogens, induces innate immune responses in plants, as well as in animals. Analogously, a nematode pheromone, the ascaroside ...ascr#18, induces hallmark plant defences including activation of (a) mitogen‐activated protein kinases, (b) salicylic acid‐ and jasmonic acid‐mediated defence signalling pathways and (c) defence gene expression and provides protection to a broad spectrum of pathogens. Ascr#18 is a member of an evolutionarily conserved family of nematode signalling molecules and is the major ascaroside secreted by plant–parasitic nematodes. Here, we report the effects of ascr#18 on resistance in four of the major economically important crops: maize, rice, wheat and soybean to some of their associated pathogens. Treatment with low nanomolar to low micromolar concentrations of ascr#18 provided from partial to strong protection in seven of eight plant–pathogen systems tested with viruses, bacteria, fungi, oomycetes and nematodes. This research may have potential to improve agricultural sustainability by reducing use of potentially harmful agrochemicals and enhance food security worldwide.
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 (IC50) = 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. PUBLICATION ABSTRACT
We explored the antifungal activity of thanatin, a 21 amino acid synthetic peptide from the hemipteran spined soldier bug Podisus maculiventris, against the mycotoxin‐producing plant pathogenic ...ascomycete Fusarium graminearum. In vitro germination assays showed complete inhibition of macroconidia germination and mycelia growth by >10 μm thanatin. Moreover, detached leaves of thanatin‐expressing Arabidopsis thaliana plants displayed enhanced resistance towards colonization with F. graminearum. Consistent with this, the plants showed also enhanced resistance of detached leaves to colonization with Botrytis cinerea. The results demonstrate a potential of thanatin for use in plant protection.
Barley microrchidia ATPases bind to DNA, have endonuclease activity, and regulate plant immunity
.
MORC1 and MORC2, two of the seven members of the Arabidopsis (
Arabidopsis thaliana
) Compromised ...Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (
Hordeum vulgare
) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of
MORC
in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (
Blumeria graminis
f. sp.
hordei
), while
MORC
overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to
Fusarium graminearum
. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn
2+
-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.
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