Mlo-based Resistance Research Articles

NLR- and mlo-Based Resistance Mechanisms against Powdery Mildew in Cannabis sativa.

Powdery mildew (PM) is one of the most common Cannabis sativa diseases. In spite of this, very few documented studies have characterized the resistance genes involved in PM defense mechanisms, or sources of natural genetic resistance in cannabis. The focus of the present work is on the two primary mechanisms for qualitative resistance against PM. The first is based on resistance (R) genes characterized by conserved nucleotide-binding site and/or leucine-rich repeat domains (NLRs). The second one involves susceptibility (S) genes, and particularly mildew resistance locus o (MLO) genes, whose loss-of-function mutations seem to be a reliable way to protect plants from PM infection. Cannabis defenses against PM are thus discussed, mainly detailing the strategies based on these two mechanisms. Emerging studies about this research topic are also reported and, based on the most significant results, a potential PM resistance model in cannabis plant-pathogen interactions is proposed. Finally, innovative approaches, based on the pyramiding of multiple R genes, as well as on genetic engineering and genome editing methods knocking out S genes, are discussed, to obtain durable PM-resistant cannabis cultivars with a broad-spectrum resistance range.

Highly effective mlo-based powdery mildew resistance in hexaploid wheat without pleiotropic effects

Application of the mlo-based resistance in barley against powdery mildew attacks is a major success in crop breeding, since it confers durable disease resistance. Resistance caused by mutations in the Mlo gene seems to be ubiquitous across a range of species. This work addresses the introduction of mlo-based resistance into hexaploid wheat, which is complicated by the occurrence of three homoeologous genes: Mlo-A1, Mlo-B1 and Mlo-D1. EMS-generated mutant plants were screened for mutations in the three homoeologues. We selected and combined 6, 8, and 4 mutations, respectively, to obtain triple homozygous mlo mutant lines. Twenty-four mutant lines showed highly effective resistance towards attack by the powdery mildew pathogen under field conditions. All 18 mutations appeared to contribute to resistance; however, they had different effects on the occurrence of symptoms such as chlorotic and necrotic spots, which are pleiotropic to the mlo-based powdery mildew resistance. We conclude that to obtain highly effective powdery mildew resistance in wheat and to avoid detrimental pleiotropic effects, all three Mlo homoeologues should be mutated; however, at least one of the mutations should be of the weaker type in order to alleviate strong pleiotropic effects from the other mutations.

CRISPR/Cas9 based mlo-mediated resistance against Podosphaera xanthii in cucumber (Cucumis sativus L.).

Powdery mildews (PM) are common and severe pathogen groups that threaten plants, and PM resistance is complex and polygenic in cucumbers. Previously mlo-based resistance was reported in various plants, including cucumber, with generated loss-of CsaMLO function mutants. However, mlo-based resistance in cucumber is also complex and involves additional mechanisms such as hypersensitive response (HR) and papillae formation. For this reason, we focused on determining the mlo-based powdery mildew resistance mechanism in cucumber. CRISPR/Cas9 was used in the present study to generate loss-of-function mutants for CsaMLO1, CsaMLO8, and CsaMLO11 of PM susceptible ADR27 cucumber inbred lines and CsaMLO mutants were obtained and validated. Trypan Blue and DAB staining were performed to detect Podosphaera xanthii germination/penetration rates and accumulation of Reactive Oxygen Species (ROS). Our results indicate that PM-susceptibility associated CsaMLOs in cucumber are negative regulators in different defense mechanisms against powdery mildew at early and late stages of infection. Further, the experiment results indicated that CsaMLO8 mutation-based resistance was associated with the pre-invasive response, while CsaMLO1 and CsaMLO11 could be negative regulators in the post-invasive defense response in cucumber against P. xanthii. Although the loss-of CsaMLO8 function confers the highest penetration resistance, CsaMLO1 and CsaMLO11 double mutations could be potential candidates for HR-based resistance against PM pathogen in cucumber. These results highlighted the crucial role of CRISPR/Cas9 to develop PM resistant cucumber cultivars, possessing strong pre-invasive defense with CsaMLO8 or post-invasive with CsaMLO1/CsaMLO11 mutations.

Gene Gun-Mediated Transient Gene Expression for Functional Studies in Plant Immunity.

One major threat to plant cultivation are fungal pathogens, which can cause substantial yield losses in agriculture. As an example, cereal powdery mildew fungi such as the barley (Hordeum vulgare) pathogen, Blumeria graminis f. sp. hordei (Bgh), are among the ten most relevant fungal plant pathogens in molecular plant pathology and can lead to yield losses of up to 30%. Plant Mildew resistance Locus O (MLO) genes are required for successful colonization of plants by powdery mildew fungi. Accordingly, loss-of-function mlo mutants confer durable resistance against powdery mildew fungi in many plant species. In the case of barley, mlo-based resistance has been used for more than 40years in agriculture without powdery mildew fungi effectively overcoming this kind of immunity. However, the molecular basis of mlo resistance and function(s) of the transmembrane Mlo protein(s) are still incompletely understood. The generation of transgenic barley plants to study the plant immune response and the involvement of Mlo therein is time-consuming and challenging. Therefore, transient gene expression via gene gun-mediated particle bombardment became a popular, easy, and efficient tool to investigate different aspects of plant defense responses in barley. Since Bgh fails to penetrate leaf epidermal cells of mlo mutants, single-cell complementation upon biolistic transformation resulting in (over-)expression of Mlo can be used to characterize the Mlo protein functionally in vivo. In this chapter, we describe in detail the gene gun-mediated transient expression of Mlo in barley leaf epidermal cells followed by powdery mildew inoculation and the subsequent microscopic evaluation. However, gene gun-mediated transient gene expression may be also used to address other research questions or to transform the epidermal tissues of other plant organs and/or species.

Europe's Farm to Fork Strategy and Its Commitment to Biotechnology and Organic Farming: Conflicting or Complementary Goals?

The European Commission's Farm to Fork (F2F) strategy, under the European Green Deal, acknowledges that innovative techniques, including biotechnology, may play a role in increasing sustainability. At the same time, organic farming will be promoted, and at least 25% of the EU's agricultural land shall be under organic farming by 2030. How can both biotechnology and organic farming be developed and promoted simultaneously to contribute to achieving the Sustainable Development Goals (SDGs)? We illustrate that achieving the SDGs benefits from the inclusion of recent innovations in biotechnology in organic farming. This requires a change in the law. Otherwise, the planned increase of organic production in the F2F strategy may result in less sustainable, not more sustainable, food systems.

Development of mlo-based resistance in tetraploid wheat against wheat powdery mildew.

Powdery mildew is a severe disease in wheat. In barley, durable resistance exists, based on non-functionality of the Mlo gene. As a model to analyse the effects of mutagenesis in the homoeologous Mlo genes of wheat, we developed mlo-based powdery mildew resistance in tetraploid durum wheat. To obtain Mlo mutations, we screened a TILLING population developed in tetraploid wheat "Kronos" for which the captured exome sequence of > 1500 lines is available. This resulted in 23 mutants for Mlo-A1 and 26 non-redundant mutants for Mlo-B1. Two Mlo-A1 and four Mlo-B1 mutants were crossed to obtain eight F2 mutant lines that showed a range of phenotypes from susceptibility to full resistance. Pot experiments under semi-field conditions confirmed the resistance levels for six of the mutants without any signs of adverse pleiotropic effects. Resistance ranking was similar across six powdery mildew isolates, indicating no isolate specificity of the mlo-based resistance. The effect of mutations in the Mlo-B1 gene was stronger than in the Mlo-A1 gene, probably reflecting differences in wild-type Mlo gene expression levels. Strong partial resistance effects were observed with single mlo-B1 mutations hence, revealing a dosage effect of mlo mutant alleles. Two of the four mlo-B1 mutations (W163* and P335L) were very strong; however, the highest combined effect was observed with the MloA-P335S/MloB-P335L combination, suggesting that non-functional, but full-length Mlo proteins might have the strongest effect compared with nonsense mutations. Our results show that mlo-based resistance might offer possibilities to introduce durable protection in tetraploid wheat against powdery mildew.

Chemical suppressors of mlo-mediated powdery mildew resistance.

Loss-of-function of barley mildew locus o (Mlo) confers durable broad-spectrum penetration resistance to the barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). Given the importance of mlo mutants in agriculture, surprisingly few molecular components have been identified to be required for this type of resistance in barley. With the aim to identify novel cellular factors contributing to mlo-based resistance, we devised a pharmacological inhibitor screen. Of the 41 rationally chosen compounds tested, five caused a partial suppression of mlo resistance in barley, indicated by increased levels of Bgh host cell entry. These chemicals comprise brefeldin A (BFA), 2′,3′-dideoxyadenosine (DDA), 2-deoxy-d-glucose, spermidine, and 1-aminobenzotriazole. Further inhibitor analysis corroborated a key role for both anterograde and retrograde endomembrane trafficking in mlo resistance. In addition, all four ribonucleosides, some ribonucleoside derivatives, two of the five nucleobases (guanine and uracil), some guanine derivatives as well as various polyamines partially suppress mlo resistance in barley via yet unknown mechanisms. Most of the chemicals identified to be effective in partially relieving mlo resistance in barley also to some extent compromised powdery mildew resistance in an Arabidopsis mlo2 mlo6 double mutant. In summary, our study identified novel suppressors of mlo resistance that may serve as valuable probes to unravel further the molecular processes underlying this unusual type of disease resistance.

Mlo-Based Resistance: An Apparently Universal "Weapon" to Defeat Powdery Mildew Disease.

Loss-of-function mutations of one or more of the appropriate Mildew resistance locus o (Mlo) genes are an apparently reliable "weapon" to protect plants from infection by powdery mildew fungi, as they confer durable broad-spectrum resistance. Originally detected as a natural mutation in an Ethiopian barley landrace, this so-called mlo-based resistance has been successfully employed in European barley agriculture for nearly four decades. More recently, mlo-mediated resistance was discovered to be inducible in virtually every plant species of economic or scientific relevance. By now, mlo resistance has been found (as natural mutants) or generated (by induced mutagenesis, gene silencing, and targeted or nontargeted gene knock-out) in a broad range of monocotyledonous and dicotyledonous plant species. Here, we review features of mlo resistance in barley, discuss approaches to identify the appropriate Mlo gene targets to induce mlo-based resistance, and consider the issue of pleiotropic effects often associated with mlo-mediated immunity, which can harm plant yield and quality. We portray mlo-based resistance as an apparently universal and effective weapon to defeat powdery mildew disease in a multitude of plant species.

TARGETED MUTAGENESIS OF MLO-HOMOLOGOUS GENES IN THE ROSE GENOME

Powdery mildew caused by Podosphaera pannosa is one of the most severe diseases of roses. Especially for greenhouse production of cut roses it depicts a significant problem. Biology and race structure of P. pannosa limit the effectiveness of race-specific, monogenic resistances making the application of fungicides indispensable. However, this application is not very desirable because of high costs, increasing legal restrictions and public concerns. An alternative to overcome this problem can be the mildew resistance locus o (mlo)-mediated resistance. The mlo-based resistance has been characterized in barley, A. thaliana, tomato and pea where the loss-of-function of specific members of the MLO gene-family leads to a recessive and durable broad-spectrum resistance. In rose four MLO homologs (RhMLO1, RhMLO2, RhMLO3 and RhMLO4) closely related to the functional ones in A. thaliana, pea and tomato have been sequenced and mapped. The aim of this work is a reverse genetics approach to verify the functionality of the four mentioned rose MLOs regarding the mediation of susceptibility towards P. pannosa. Therefore, stable transgenics of the tetraploid Rosa hybrid cultivar ‘Pariser Charme’ expressing an RNAi construct for all four genes are produced. Several shoots could be regenerated and transferred to soil and first analyses to determine the transgenic character of the plants have been performed.

Antisense RhMLO1 Gene Transformation Enhances Resistance to the Powdery Mildew Pathogen in Rosa multiflora

Powdery mildew is one of the most important crop diseases worldwide. Genetic analysis has revealed that mutant alleles of the Mlo gene cause broad-spectrum resistance against pathogens found in cereal. In this study, the possibility of inducing powdery mildew (Podosphaera pannosa) resistance via transgenic technology in the rose was examined. The transgenic lines were confirmed for integration and copy number of the transgenes based on PCR and Southern blots. A clear correlation was found between resistance and the accumulation of silenced RhMLO1, based on real-time fluorescent quantitative PCR and resistance analysis. Compared with the control plants, transgenic roses displayed higher resistance levels. Furthermore, the results indicated that RhMLO1 has a negative role in the rose–powdery mildew pathogen interaction. This demonstrates the potentially viable strategy of rendering the Mlo homologs partially non-functional via a transgenic approach or mutagenesis in order to counter powdery mildew in Rosa. To the best of our knowledge, this study is one of the first attempts at using a Mlo-based resistance strategy to combat powdery mildew in Rosa.

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.