Loss Of Function Mutations In a Cucumber Mlo Gene Lead To Hypocotyl Resistance To Powdery Mildew

Loss-of-function Mutations in a Cucumber MLO Gene Lead to Hypocotyl Resistance to Powdery Mildew

Jeroen A. Berg*, Yuling Bai, and Henk J. Schouten

Wageningen UR Plant Breeding, Wageningen University & Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. e-mail: [anonimizat]

Abstract

Powdery mildew (PM) is an important disease of cucumber (Cucumis sativus). We are interested in the identification of susceptibility genes in cucumber. Mutations leading to loss of function or silencing of susceptibility genes can lead to durable, broad range resistance against PM. CsaMLO8, a cucumber homolog of the well-known barley MLO gene, was previously identified as a candidate susceptibility gene for PM in cucumber. MLO genes form gene families, in which two clades have previously been shown to harbour susceptibility genes: clade IV in monocot species and clade V in dicot species. CsaMLO8 belongs to clade V and co-localizes with a QTL on chromosome 5 for hypocotyl-specific resistance to PM in cucumber. Recently, two research groups published about the functional characterization of CsaMLO8 and the identification of mutant alleles of this gene. In this review we compare the findings from these two groups, and discuss the current knowledge about MLO genes in PM susceptibility.

Keywords: Cucumber, powdery Mildew, MLO genes, hypocotyl resistance

Powdery mildew resistance in cucumber

With an annual worldwide production value of over 33 billion US dollar, cucumber (Cucumis sativus L.) is an economically important crop (FaoStat 2016). The genome of cucumber (‘Chinese Long’ inbred line 9930) was sequenced as one of the first vegetable crops, and is publicly available since 2009 (Huang et al. 2009). Furthermore, several cucumber accessions have been resequenced (Woycicki et al. 2011; Qi et al. 2013). This wealth of information can make cucumber a model species for other cucurbit crops, such as melon, watermelon and pumpkin.

Powdery mildew (PM) is a devastating fungal disease in many crop species including cucurbits such as cucumber. Two species of fungi have been reported to cause PM in cucumber, Podosphaera xanthii (synonymous with P. fusca, previously named Sphaerotheca fuliginea) and Golovinomyces cichoracearum (previously named Erysiphe cichoracearum). In recent decades, P. xanthii is considered to be the main causal agent of PM in cucurbits, especially under greenhouse conditions (Block and Reitsma 2005; Perez-Garcia et al. 2009). Breeding of resistant cucumber varieties has been undertaken for several decades (for example, Shanmugasundaram et al. 1971; Sitterly 1972), but underlying resistance genes have to date not been functionally characterised.

Several sources of resistance to PM in cucumber have been identified, especially from Asia, such as the Indian accessions PI 197087 and PI 197088 and the Japanese ‘Natsufushinari’ (Kooistra 1968). Resistance in those materials was shown to be under the control of multiple genes, most of which show recessive inheritance (Kooistra 1968). Shanmugasundaram et al. (1971) showed that one major recessive gene, s, was the most important factor contributing to powdery mildew resistance. This recessive gene s confers intermediate or hypocotyl resistance, characterised by completely resistant hypocotyl, stem and petiole tissue and partially resistant leaves and cotyledons. Combinations of this gene with other sources of resistance led to full resistance (Shanmugasundaram et al. 1971). In a more recent study, He et al. (2013) mapped several QTLs for PM resistance, scoring resistance of hypocotyl, cotyledon and true leaf tissue separately. The authors reported three QTLs for hypocotyl resistance, one with a major effect and two with a minor effect. They conclude that the major effect QTL, pm5.2, is probably the same as the gene s described by Shanmugasundaram et al. (1971).

Introgression of dominantly inherited resistance (R) genes, usually from wild relatives is the traditional approach to obtain resistance in a crop. R genes encode proteins, often of the nucleotide-binding, leucine-rich-repeat (NB-LRR) type, which are able to recognise a pathogen and trigger a strong defence response, usually associated with a hypersensitive response (HR) leading to cell death. The R gene product either directly recognises corresponding avirulence (Avr) gene products of the pathogen, or modifications of host factors by Avr gene products (Jones and Dangl 2006). The direct relation of plant R genes with their cognate Avr genes in the pathogen is known as the gene-for-gene relationship (Flor 1971). Whereas introgression of a new R gene initially gives good resistance against a pathogen, it puts selective pressure on the pathogen to evolve the corresponding Avr gene in such a way that it is no longer recognised by the host plant. Therefore, R-gene based resistance is often breached by new, virulent races of the pathogen quite soon, especially for versatile pathogens, such as powdery mildew fungi (Jones and Dangl 2006). The sequenced genome of cucumber harbours only 61 NB-LRR genes (Huang et al. 2009), a surprisingly low number compared to, for example, the 149 NB-LRR genes in the model plant Arabidopsis thaliana (Meyers et al. 2003), 500 in rice (Monosi et al. 2004) or even 1015 in apple (Arya et al. 2014). Similar low numbers have been reported in other cucurbit taxa, such as 44 NB-LRR genes in watermelon (Guo et al. 2013) and 81 in melon (Garcia-Mas et al. 2012).

During the last decades, a novel alternative for R-gene mediated resistance has grown in popularity, which is the identification of impaired susceptibility (S) genes (Vogel and Somerville 2000; Eckardt 2002; Pavan et al. 2010; van Schie and Takken 2014). In contrast to R genes recognising a pathogen, S genes are plant genes facilitating compatible interactions between the plant host and its pathogen, for instance by negative regulation of defence responses, facilitating host recognition by the pathogen, or being essential for the uptake of nutrients by the pathogen (van Schie and Takken 2014). The fact that most pathogens are able to infect only a limited amount of plant species suggests that there must be specific genes in those plant species that are essential for the pathogen. This is especially true for biotrophic pathogens such as mildew-causing fungi taxa, which rely on a long-lasting interaction with living host cells (Eckardt 2002). Loss-of-function mutations in an S gene are thought to lead to durable, broad-spectrum, recessively inherited resistance (Eckardt 2002; Pavan et al. 2010). As inheritance of resistance against powdery and downy mildews in cucumber has often been reported to be recessive (Kooistra 1968; Shanmugasundaram et al. 1971; van Vliet & Meijsing 1977), it is likely that loss-of-function mutations in S genes rather than (dominantly inherited) R genes are causal to many known sources of resistance in cucumber.

MLO susceptibility genes

One of the oldest and best-known examples of impaired S genes is the MLO gene family. Mutant alleles of the “Mildew Locus O” (mlo) were originally found in the 1940s in mutagenized barley populations (Freisleben and Lein 1942), which became resistant against Blumeria graminis f.sp. hordei, the causal agent of PM in barley. Barley varieties with mlo resistance have since been grown in the field for several decades without breaching of resistance by virulent new mildew races to date, providing evidence for the durability of S-gene based resistance (Jorgensen 1992). After the barley MLO gene was cloned (Büschges et al. 1997), it was found that MLO genes are conserved throughout the plant kingdom and occur in plants as a multi-copy gene family (Devoto et al. 1999; Devoto et al. 2003). Recently, Kusch et al. (2016) provided evidence for the occurrence of MLO-like genes in representatives of all land plants, including mosses and gymnosperms, in related unicellular algae, and even in distantly related eukaryotes such as Amoebozoa and Chromalveolata, the latter intriguingly including plant pathogens such as Phytophtora infestans and Hyaloperonospora arabidopsidis.

In several plants, some, but not all, MLO genes have been found to be involved in PM susceptibility, such as Arabidopsis, tomato, pea, pepper, tobacco, bread wheat and potentially also grapevine and peach (Consonni et al. 2006; Bai et al. 2008; Feechan et al. 2008; Jiwan et al. 2013; Zheng et al. 2013; Ning et al. 2014; Appiano et al. 2015a). It has been found that in phylogenetic trees of the MLO gene family, all S-genes for PM cluster in two clades, namely clade IV for S-genes in monocot species and clade V for dicot species. The other five clades harbour MLO-like genes that have not been found to be S-genes (Devoto et al. 2003; Kusch et al. 2016).

Recently, Appiano et al. (2015b) showed that despite the occurrence of clade-specific molecular features, overexpression of a clade IV MLO gene (i.e. HvMLO) was able to restore susceptibility in a tomato mutant that is PM resistant because of a loss-of-function mutation in a Clade V MLO gene (SlMLO1). This proves that clade IV and clade V MLO genes are functionally conserved in the plant-pathogen interaction. Additionally, amino acid substitutions in MLO genes leading to PM resistance in either the monocot barley or the dicot Arabidopsis were reviewed, and it was found that amino acids required for susceptibility were usually conserved between clade IV and clade V MLO genes (Appiano et al. 2015b). In addition to single nucleotide variants leading to amino acid substitutions, other types of natural loss-of-function mutations have been identified in MLO genes. In tomato, a 19 bp frameshift deletion in the gene SlMLO1 was found to be causal to recessive ol-2 resistance (Bai et al. 2008). In pea, several allelic variants of the MLO gene PsMLO1 were found to be causal to the er1 locus, including small deletions and a transposable element insertion leading to aberrant splicing at the transcript level (Humphry et al. 2011). In the PM resistant tobacco ‘Kobuku’, nucleotide substitutions were identified in splice sites of introns of both NtMLO1 and NtMLO2 genes, leading to aberrant splicing of both genes at the transcript level (Fujimura et al. 2016).

With the cucumber genome sequence publicly available, two groups searched for MLO homologs in cucumber. Zhou et al. (2013) reported that they identified 14 MLO-like genes in the first version of the cucumber genome. Schouten et al. (2014) used the updated version of the cucumber genome (v2) and identified 13 MLO-like genes, the difference being that in the first version of the genome one MLO gene on chromosome 1 was erroneously annotated as two shorter neighbouring genes. Both groups agree that three cucumber MLO genes cluster in clade V of the MLO gene family, and can therefore be considered to be candidate S-genes. In the nomenclature proposed by Schouten et al. (2014), which we shall follow in this article, the names of those clade V MLO genes are CsaMLO1, CsaMLO8 and CsaMLO11.

Recently, MLO-like genes were also identified in other cucurbit crops, 14 MLO in watermelon, 16 in melon and 18 in pumpkin (Iovieno et al. 2015). However, it should be noted that the lists of melon and pumpkin homologs contain several very short genes, some of which are adjacent to one another. Potentially those are errors in gene prediction, reflecting the poor quality of the current genome annotations for these species, so the actual number of MLO genes could be lower, reminiscent of the case in cucumber where at first 14 homologs were identified due to annotation problems. In melon and watermelon, three clade V MLO genes were identified, versus four clade V MLO genes for pumpkin. In the phylogenetic analysis, three orthologous groups of cucurbit clade V MLO genes can be distinguished (Iovieno et al. 2015), indicating that the last common ancestor of cucurbits probably already had three clade V MLO genes, with one additional gene duplication in the branch leading to pumpkin.

CsaMLO8 is a cucumber S-gene for PM

Interestingly, one of the three clade V MLO genes identified in cucumber, CsaMLO8, is located on the interval of a major QTL for hypocotyl resistance to PM described by He et al. (2013). This led us to the hypothesis that a mutation in this MLO gene might be causal to the hypocotyl resistance that was reported more than 40 years ago (Shanmugasundaram et al. 1971), and which is still frequently used in cucumber breeding, indicating the durability of this resistance. In our recent publication (Berg et al. 2015), we reported the cloning and functional characterization of CsaMLO8 from both susceptible and (hypocotyl) resistant cucumber genotypes. Simultaneously, another group described the fine-mapping of a major PM-resistance QTL from a highly resistant North China type cucumber inbred line to a 170 kb interval on chromosome 5 containing 25 genes, one of which was CsaMLO8 (Nie et al. 2015a). They subsequently characterized this gene, to which they refer in their publication as CsMLO1, the results of which confirmed our analysis and added additional information (Nie et al. 2015b). In this review we compare the findings of the two groups, summarizing the current knowledge about CsaMLO8.

Both Berg et al. (2015) and Nie et al. (2015b) cloned the coding sequence of CsaMLO8 from cDNA of susceptible cucumber genotypes, and confirmed the predicted wild-type sequence of CsaMLO8. Berg et al. (2015) overexpressed CsaMLO8 in a tomato mutant that is PM resistant because of a loss-of-function mutation in a Clade V MLO gene (SlMLO1). Complementing this mutant with the wild-type CsaMLO8 gene from cucumber restored susceptibility in six out of ten transformants. Nie et al. (2015b) overexpressed CsaMLO8 in PM-resistant double atmlo2/atmlo12 Arabidopsis mutants, and observed restoration of susceptibility in seven transformants. The results of both groups are in agreement with each other. The fact that CsaMLO8 is able to functionally complement loss-of-function mutations in unrelated species strengthens the conclusion that CsaMLO8 is a functional susceptibility gene for PM.

Both research groups studied the expression of CsaMLO8 in various cucumber tissues, although the experimental set-ups were different from each other. Berg et al. (2015) quantified the expression of CsaMLO8 in leaf, hypocotyl and cotyledon tissue before and at several time-points after inoculation with Podosphaera xanthii and found that, whereas in the hypocotyl the expression significantly increased at four and six hours post inoculation, the expression in leaves and cotyledons remained constant over the time course. This supported the notion that mutations in CsaMLO8 give full resistance in hypocotyl tissue, and only partial resistance in leaf tissue.

Nie et al. (2015b), however, quantified CsaMLO8 expression in more tissues, including hypocotyl, cotyledon and leaf but also root, stem, male and female flowers and fruits, without inoculating the plants with Podosphaera xanthii. They concluded that in non-inoculated plants, CsaMLO8 is highly expressed in leaves, cotyledons and flowers, and lower in hypocotyl and stem tissue. CsaMLO8 was found to be barely expressed in roots and fruits. Although it looks at first glance like the data presented by Berg et al. (2015) and by Nie et al. (2015b) contradict each other, one should notice that Berg et al. (2015) did not directly compare the expression in the different tissues with one another but instead analysed the (normalized) expression within the different tissues over a time course. Re-analysis of the data presented by Berg et al. (2015) confirms the finding by Nie et al. (2015b) that, in non-inoculated cucumber plants, CsaMLO8 is more highly expressed in leaf and cotyledonary tissue than in hypocotyl tissue. This does not exclude the finding by Berg et al. (2015) that, in hypocotyl tissue, CsaMLO8 expression is induced by PM inoculation.

Nie et al. (2015b) furthermore quantified CsaMLO8 expression in leaves of Podosphaera xanthii inoculated cucumbers over a time course, and found that CsaMLO8 expression was induced, particularly at 12 hours post inoculation. This contradicts the finding of Berg et al. (2015) that CsaMLO8 expression is not significantly induced in leaf tissue upon inoculation with powdery mildew, although it should be noted that Berg et al. (2015) did not quantify the CsaMLO8 expression at 12 hours post inoculation, so it is possible that a significant upregulation was missed.

Nie et al. (2015b) also made fusion constructs of CsaMLO8 with GFP, to observe the subcellular localization of the protein. As was expected, the CsaMLO8 protein localized to the plasma membrane.

Mutant alleles of CsaMLO8

Berg et al. (2015) cloned the coding sequence of CsaMLO8 from a hypocotyl-resistant cucumber line, and observed two different splicing variants, resulting in deletions of either 72 bp or 174 bp compared to the wild-type gene. qRT-PCR using splice junction spanning primers revealed that the 174 bp deletion product was the most abundant isoform. Sequencing of the deletion region from genomic DNA revealed the presence of a 1449 bp insertion in exon 11 of the gene. Characterisation of this insertion showed that it had long terminal repeats (LTR) with a length of 184 bp, as well as a 5 bp target site duplication (TSD). In the sequenced cucumber genome, at least 44 other insertions of near-identical sequences could be identified, leading to the conclusion that this is a family of transposable elements (TE) of the LTR type. During splicing of the pre-mRNA of CsaMLO8, the TE is apparently spliced out together with either the complete exon 11 (174 bp) or a smaller part of exon 11 (72 bp). Berg et al. (2015) overexpressed the 174 bp deletion variant of CsaMLO8 in Slmlo1 tomato, and showed that it is unable to restore susceptibility in tomato. This underlines that the TE insertion in the gene, and subsequent loss of a part of the gene at the RNA-level, led to a non-functional allele of CsaMLO8, and therewith to hypocotyl resistance.

Nie et al. (2015a) identified the exact same 1449 bp insertion in their resistant material during fine-mapping of a recessively inherited QTL for PM resistance. They reported that it leads to the loss of exon 11 (174 bp) on the cDNA level, in agreement with the findings of Berg et al. (2015). They do not report the 72 bp deletion isoform, although the picture of their gel clearly shows two bands, suggesting that in their material there are indeed two isoforms as well.

In addition to characterising this particular mutation in their resistant material, Nie et al. (2015b) amplified the genomic CsaMLO8 sequence of 27 additional cucumber inbred lines of diverse adaptation and origin, including well-known sources of PM resistance such as WI 2757 (He et al. 2013) and (descendants of) PI 197088. Whereas they found the same 1449 bp insertion allele in nine of the additional resistant cucumber lines, including WI 2757, they also characterised two additional natural variants in other resistant lines. In four instances, they found a T to C point mutation with respect to their susceptible allele at position 1301, leading to erroneous splicing of exon 5. In one case, they found a 1 bp frameshift insertion at position 3703, leading to an early stop codon. Whereas most of the susceptible lines (11 out of 14) were found to have the wild-type CsaMLO8 allele, there were some exceptions: one susceptible line, “S49”, was found to have the 1449 bp transposable element insertion, and another susceptible line, “9930”, which is the genotype of the cucumber reference genome, was found to have the T to C point mutation leading to aberrant splicing of exon 5. Apparently, in these two inbred lines, the CsaMLO8 mutation is not sufficient for obtaining resistance, which might be explained by the fact that hypocotyl resistance is known to be only partially effective (Shanmugasundaram et al. 1971), so having the CsaMLO8 mutation in a particularly susceptible background does not lead to resistance. Alternatively, it could be possible that those genotypes have mutated alleles of genes required for mlo resistance, like the ror genes described in barley (Freialdenhoven et al. 1996) and pen genes in Arabidopsis (Consonni et al. 2006).

Interestingly, another susceptible line was found to have a 1451 bp insertion with a sequence very similar (95.6% identical) to the 1449 bp insertion, but at a different location, intron 9 instead of exon 11. This insertion in the intron was found not to alter the coding sequence nor the transcript abundance of CsaMLO8, explaining the susceptibility of this genotype. However, it indicates that insertions of this TE did occur frequently in the cucumber genome.

Berg et al. (2015) did not attempt sequencing CsaMLO8 alleles from other cucumber genotypes. Instead they examined the occurrence of the 1449 bp insertion in the publicly available dataset of 115 resequenced cucumber genotypes (Qi et al. 2013), and found that the TE-allele of CsaMLO8 occurred frequently, in 31 out of the 115 genotypes. This is in accordance with the fact that Nie et al. (2015b) found this allele to be the most common among resistant genotypes, and indicates that it has been actively selected for during cucumber breeding. One of the 31 genotypes with the TE-allele was found to be a semi-wild, Cucumis sativus var. hardwickii (Royle) Alef. accession. Possibly, a C. sativus var. hardwickii accession has been the donor of this allele, introgressed into commercial cultivars.

For the current review, we tried to identify lines among the 115 resequenced cucumber genotypes (Qi et al. 2013) with the other loss-of-function alleles discovered by Nie et al. (2015b). For the T to C point mutation at position 1301 described by Nie et al. (2015b), we should note that as pointed out by the authors, the reference genome “9930” has this mutation. We could identify three other genotypes homozygous for a C at this position, and additionally three heterozygous genotypes (Table 1), confirming the finding by Nie et al. (2015b) that this allele is less common than the 1449 bp insertion. Interestingly, one of the three genotypes homozygous for this allele originated from Puerto Rico, and might therefore be related to accessions Puerto Rico 37 and Puerto Rico 40, the first reported sources of resistance to PM in cucumber (Kooistra 1968). We could not identify any lines with the one bp insertion at position 3703 reported by Nie et al. (2015b), which is remarkable as the accession in which Nie et al. (2015b) identified this mutation, PI 197088, is a well-known and often-used source of resistance to both powdery and downy mildew.

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Table 1. Cucumber genotypes among the 115 lines resequenced by Qi et al. (2013) homozygous or heterozygous for the CsaMLO8 T to C point mutation at position 1301.

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Conclusions

Summarizing, the combined works of Berg et al. (2015) and Nie et al. (2015b) show that CsaMLO8 is a functional susceptibility gene in cucumber, and that several natural loss-of-function mutations of this susceptibility gene have been used in breeding to obtain powdery mildew resistance. The most commonly occurring mutation of this gene in cucumber breeding material is an insertion of a 1449 bp transposable element in exon 11. The expression of CsaMLO8 is highest in leaves, cotyledons and flowers, and lower in hypocotyl and stem tissue. However, inoculation with powdery mildew induces expression of CsaMLO8 in the hypocotyl. Whether or not CsaMLO8 expression is also induced in leaf tissue is still under debate.

The mutant alleles of CsaMLO8 are to our knowledge the first known example of impaired S-gene alleles in a cucurbit species. As resistances in cucurbits have previously often shown to inherit recessively, and cucurbits have a low number of NB-LRR genes, it can be expected that in the future more impaired susceptibility genes in cucurbits will be discovered. MLO-like genes have also been identified in melon, watermelon and pumpkin (Iovieno et al. 2015). The information presented about CsaMLO8 might be useful in identifying orthologous susceptibility genes in those other cucurbit crops.

Acknowledgements

The authors are grateful for funding from foundation TKI Starting Materials and Bayer Vegetable Seeds.

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