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Published: 04 July 2024

The ACE1 secondary metabolite gene cluster is a pathogenicity factor of wheat blast fungus

Communications Biology volume 7, Article number: 812 (2024) Cite this article

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Abstract

Wheat blast caused by Pyricularia oryzae pathotype Triticum is now becoming a very serious threat to global food security. Here, we report an essential pathogenicity factor of the wheat blast fungus that is recognized and may be targeted by a rice resistance gene. Map-based cloning of Pwt2 showed that its functional allele is the ACE1 secondary metabolite gene cluster of the wheat blast fungus required for its efficient penetration of wheat cell walls. ACE1 is required for the strong aggressiveness of Triticum, Eleusine, and Lolium pathotypes on their respective hosts, but not for that of Oryza and Setaria pathotypes on rice and foxtail millet, respectively. All ACE1 alleles found in wheat blast population are recognized by a rice resistance gene, Pi33, when introduced into rice blast isolates. ACE1 mutations for evading the recognition by Pi33 do not affect the aggressiveness of the rice blast fungus on rice but inevitably impair the aggressiveness of the wheat blast fungus on wheat. These results suggest that a blast resistance gene already defeated in rice may be revived as a durable resistance gene in wheat by targeting an Achilles heel of the wheat blast fungus.

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Introduction

We are now threatened not only by a human pandemic disease but also by a plant disease that is becoming pandemic¹. Wheat blast, a destructive disease of our staple crop, first emerged in Brazil in 1985². It subsequently spread to neighboring countries, i.e., Bolivia in 1996, Paraguay in 2002, Argentina in 2007, and became a major threat to wheat production in South America³. Its causal agent is a subgroup of Pyricularia oryzae (syn. Magnaporthe oryzae)⁴, i.e., the Triticum pathotype (MoT)⁵ which evolved through host jumps in 1980s⁶. MoT (the wheat blast fungus) spread across continents from South America to Asia and Africa independently¹, and caused wheat blast outbreaks in Bangladesh (in 2016)^7,8 and in Zambia (in 2018)⁹. To mitigate this serious disease, resistant cultivars are needed. However, resistance genes effective against wheat blast are rarely found in wheat germplasms probably because, until recently, wheat has had no interaction with MoT. Some resistance genes or resources have been found in wheat and its relatives, e.g., Rmg8¹⁰ in common wheat and a 2NS chromosomal segment derived from Aegilops ventricosa¹¹. However, the current wheat blast population in South America already contains isolates that have almost overcome the Rmg8 and 2NS resistance^11,12,13.

In the history of breeding for resistance, most of resistance genes introduced into elite varieties have been overcome by new races and rendered ineffective soon after their releases to farmer’s fields. The ultimate goal of plant breeders struggling against plant diseases is to develop cultivars with durable resistance¹⁴. A big challenge for them is how to predict durability of resistance genes before starting breeding programs¹⁵. Resistance genes are most likely to be durable if their corresponding virulence is costly^14,16 or if their corresponding avirulence genes encode Achilles heel effectors essential for virulence^17,18. It is, therefore, strongly desired to find pathogen genes essential for virulence or pathogenicity and resistance genes targeting their products.

P. oryzae is composed of several host genus-specific pathotypes such as Oryza pathotype (MoO) pathogenic on rice, Setaria pathotype (MoS) pathogenic on foxtail millet, Eleusine pathotype (MoE) pathogenic on finger millet, and Lolium pathotype (MoL) pathogenic on perennial ryegrass^19,20, in addition to the newly evolved MoT mentioned above. These pathotypes almost correspond to distinct lineages deduced from phylogenetic analyses²¹. Through genetic analyses involving crosses between wheat (MoT) and non-wheat (MoO and MoS) pathotypes, we have previously identified three loci, Pwt1, Pwt2, and Pwt5, conditioning their specificity on wheat^22,23. The avirulence/virulence alleles at these loci were designated as PWT1/pwt1, PWT2/pwt2, and PWT5/pwt5, based on a hypothesis that the avirulence alleles were functional^23,24. Among the three loci, however, Pwt2 was associated with a peculiar phenotype; under the absence of PWT1 and PWT5, PWT2 alleles from an MoO isolate (PO12-7301-2) and an MoS isolate (GFSI1-7-2) produced discrete green lesions (designated as G−) while the pwt2 allele from an MoT isolate (Br48) produced confluent green lesions (designated as G+)^22,23. The PWT2 phenotype may be better described as “virulent but weakly aggressive” rather than avirulent. The phenotype produced by carriers of PWT2 alone was temperature-sensitive; it was indistinguishable from the completely avirulent phenotype at 20 °C, became visible as G− at 22–25 °C, and turned indistinguishable from the completely virulent phenotype at 27–28 °C^22,23,24. Cytologically, the discrete lesion with the PWT2 allele was associated with the higher frequency of papilla formation at penetration sites leading to reduced level of successful host invasion, while the confluent lesion with the pwt2 allele was associated with the reduced frequency of papilla formation^22,23. These observations suggest that, regarding the function of Pwt2, another hypothesis is possible; the functional allele at the Pwt2 locus may be pwt2 (derived from MoT) which actively suppresses the penetration defense (papilla formation) of wheat.

In the present study, we found that the functional allele at the Pwt2 locus was pwt2 derived from Br48 (MoT). The pwt2 allele was a fungal secondary metabolite gene cluster known as the ACE1 cluster^25,26. It was involved in the suppression of papilla formation, and essential for the complete pathogenicity with strong aggressiveness of MoT on wheat. In addition, we found that a blast resistance gene, Pi33, in rice could recognize ACE1 alleles derived from MoT and confer resistance. A point mutation inactivating the enzymatic activity of ACE1²⁵ in MoT for evading the recognition by Pi33 inevitably caused critical reduction of its aggressiveness on wheat. These results suggest that ACE1 and Pi33 may be an Achilles heel of MoT and an arrow targeting it. Its implications in the breeding for durable resistance are discussed here.

Results

Pwt2 is a secondary metabolite gene cluster essential for the strong aggressiveness of MoT on wheat

There are two styles of usage of terms for pathogenicity. In one style virulence is defined as the degree of pathogenicity of a given pathogen²⁷, and the term aggressiveness is rejected²⁸. In the other style virulence/avirulence is defined as the ability/inability of a pathogen to cause a compatible reaction on a host cultivar with genetic resistance while aggressiveness is defined as the amount of disease caused by an isolate of the pathogen²⁹. In the present article, we will use these terms in accordance with the latter usage.

On primary leaves of wheat cv. Norin 4 (N4), PO12-7301-2 (PWT1; PWT2; PWT5) shows avirulence with the B- phenotype (discrete brown lesions) while Br48 (pwt1; pwt2; pwt5) shows virulence with the G+ phenotype (confluent green lesions)²³. Out of the F₁ cultures derived from PO12-7301-2 x Br48, we chose 54N1 (pwt1; PWT2; pwt5) showing the G- phenotype (discrete green lesions) peculiar to cultures with PWT2 alone (virulent with weak aggressiveness) (Fig. 1d), backcrossed it with Br48 (virulent with strong aggressiveness), and produced 319 BC₁F₁ cultures (Fig. 1a). Molecular mapping suggested that the Pwt2 locus was located on a region on chromosome 2 flanked by two markers, 613k_X and 488k_S (Fig. 1b). Sequence analyses in this region indicated that PO12-7301-2 did not carry additional genes compared with Br48, but lacked some genes harbored by Br48 (Fig. 1c). This result led us to an assumption that the functional allele at the Pwt2 locus may not be PWT2 derived from PO12-7301-2 but pwt2 derived from Br48. To confirm this assumption, we screened a BAC library of Br48, selected two clones containing a part or most of this region, and introduced them into 54N1. Resulting transformants showed virulence with strong aggressiveness (G+) on N4 (Fig. 1c, Supplementary Fig. 1), indicating that Pwt2 is a locus conditioning aggressiveness.

Fig. 1: *Pwt2* is a locus conditioning aggressiveness and containing avirulence gene *ACE1.*

In the region covered by the shorter clone (ZT1-1-A), we found an allele of ACE1, which had been known to control avirulence of MoO isolates on a rice resistance gene, Pi33²⁵. The ACE1 allele in Br48 (ACE1^Br48) had a complete ORF while its allele in PO12-7301-2 had a mutation leading to a stop codon (Fig. 1c). A fragment (~15 kb) containing ACE1^Br48 was subcloned from ZT1-1-A to a plasmid vector, and the resulting plasmid (pACE1^Br48) was introduced into 54N1. The transformants carrying pACE1^Br48 gained the strong aggressiveness comparable to Br48 (Fig. 1c, d). Furthermore, a disruption of ACE1^Br48 in Br48 impaired its aggressiveness, resulting in the G− phenotype, while a reintroduction of ACE1^Br48 to the disruptant had it recover the strong aggressiveness (Fig. 1e and Supplementary Fig. 2). These results suggest that the core, functional gene at the Pwt2 locus is ACE1^Br48. GFSI1-7-2, another PWT2 carrier, lacked most of the genes including ACE1 in the candidate region (Fig. 1c).

Barley has been considered to be a common host of P. oryzae although its susceptibility to P. oryzae varies according to isolates and cultivars tested³⁰. Barley cv. H.E.S.4 showed the G− and G+ phenotypes with 54N1 and Br48, respectively, as N4 did (Supplementary Table 1). In the BC₁F₁ population derived from 54N1 x Br48, G− and G+ cultures segregated in a 1:1 ratio on H.E.S.4, and the segregation pattern was perfectly concordant with that on N4 (Supplementary Table 1). Furthermore, the disruption of ACE1^Br48 in Br48 and the reintroduction of ACE1^Br48 to the disruptant resulted in the reduction and regain, respectively, of aggressiveness on H.E.S.4 as on N4 (Fig. 1f). Similar results were obtained not only at 22 °C (Fig. 1e, f) but also at 26 °C (Supplementary Fig. 3). These results suggest that ACE1^Br48 is involved in the aggressiveness of MoT on both wheat and barley.

ACE1 encodes a hybrid between a polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS)²⁵, and belongs to a cluster of 15 secondary metabolism genes (ACE1 cluster)²⁶ (Fig. 1c). The ACE1 cluster is divided into Part A including ACE1 and Part B including SYN2 encoding another PKS-NRPS³¹ (Supplementary Fig. 4). Deletion of SYN2^Br48 (an allele of SYN2 in Br48) reduced its aggressiveness on N4 to an intermediate level, and reintroduction of SYN2^Br48 to the deletion mutant had it regain the strong aggressiveness (Supplementary Fig. 4a, b). Furthermore, deletion of the whole cluster in Br48 followed by the reintroduction of its various portions suggested that the entire cluster is required for conferring the strong aggressiveness on N4 (Supplementary Figs. 4a, c, and 5). Taken together, we conclude that Pwt2 is the ACE1 cluster and is involved in the strong aggressiveness of Br48 (MoT). The ACE1 cluster of Br48 (Triticum isolate) contained all of the genes designated in the ACE1 cluster of Guy11 (Oryza isolate), and each of the Br48 genes was 99.5–100% identical to its corresponding Guy11 gene at the nucleotide and amino acid sequence levels (Supplementary Fig. 6).

ACE1 ^Br48 is involved in the suppression of the formation of fluorescent papilla

To elucidate why the loss of function of ACE1^Br48 leads to the G− phenotype, primary leaves of N4 were inoculated with Br48, its ACE1-knockout mutants (Br48ΔACE1), and transformants of Br48ΔACE1 carrying pACE1^Br48 (Br48ΔACE1 + ACE1^Br48). In leaves inoculated with Br48, more than 70% of germlings successfully invaded epidermal cells and extended infection hyphae (Fig. 2a, d). The incidence of fluorescent papillae was very low (Fig. 2b). By contrast, in those inoculated with Br48ΔACE1, more than 70% of germlings induced fluorescent papillae and failed to penetrate cell walls (Fig. 2b, e). However, a small proportion of germlings of Br48ΔACE1 evaded this reaction and penetrated cell walls (Fig. 2a). Once they entered the cells, they produced infection hyphae without inducing hypersensitive reactions, and extended the infection hyphae to the neighboring cells (Fig. 2c, f), which resulted in the peculiar phenotype, G− (discrete green lesions). In leaves inoculated with Br48ΔACE1 + ACE1^Br48, the incidence of fluorescent papillae was suppressed, and many germlings successfully invaded epidermal cells (Fig. 2a, b). Taken together, cytological analyses with those disruptants and revertants suggested that the function of ACE1^Br48 is associated with suppression of the formation of fluorescent papillae²² which inhibits cell wall penetration.

**Fig. 2: *ACE1*-knockout mutants are attenuated in penetration of wheat epidermal cell walls.**

Contribution of the ACE1 cluster to aggressiveness of P. oryzae pathotypes is dependent on host species they attack

Why could PO12-7301-2 (MoO) and GFSI1-7-2 (MoS), wild isolates lacking the functional ACE1 or ACE1 cluster, have survived in nature before isolation from natural fields? To answer this question, we first compared amino acid sequences of the ACE1 cluster in wild isolates of various pathotypes (Fig. 3a). In MoT, MoL, and MoE all isolates carried ACE1 alleles with highly conserved sequences. On the other hand, MoO and MoS contained some isolates that carried ACE1 alleles with many mutations or lacked several cluster genes. We chose one isolate carrying an intact ACE1 cluster from each pathotype (Ken53-33 from MoO, IN77-20-1-1 from MoS, MZ5-1-6 from MoE, and TP2 from MoL) and disrupted their ACE1 alleles. On barley, the aggressiveness of the four wild isolates was all impaired by the disruption (Fig. 3b–e and Supplementary Fig. 7), suggesting that their ACE1 alleles are functional and required for their aggressiveness on the common host. On their original hosts, however, the contribution of the ACE1 alleles varied depending on host species. The aggressiveness of MZ5-1-6 (MoE) and TP2 (MoL) on finger millet and perennial ryegrass, respectively, was impaired by the disruption of their ACE1 alleles (Fig. 3d, e and Supplementary Fig. 7). By contrast, the disruption of ACE1 alleles of Ken53-33 (MoO) and IN77-20-1-1 (MoS) showed no effect on their aggressiveness on rice and foxtail millet, respectively (Fig. 3b, c, and Supplementary Fig. 7). These results suggest that the ACE1 alleles are required for the infection of finger millet and perennial ryegrass, but not required for the infection of rice and foxtail millet. This is in accordance with a previous report that ACE1 is not required for pathogenicity of MoO isolates on rice²⁵. The results shown in Fig. 3b and c explain why MoO and MoS contained some isolates with mutated ACE1 alleles or those lacking several cluster genes (Fig. 3a), and provide an answer to the question why PO12-7301-2 and GFSI1-7-2 could have survived in nature.

**Fig. 3: Conservation of the *ACE1* cluster in *P. oryzae* pathotypes correlates with its significance for their aggressiveness on host species.**

ACE1 alleles of MoT isolates are recognized by rice resistance gene Pi33

ACE1 is an avirulence gene corresponding to Pi33 in the MoO-rice pathosystem²⁵, and its alleles are widely distributed in the MoT population³². If these alleles in MoT are recognized by Pi33, it may be possible to use Pi33 as a gene for resistance to wheat blast. We surveyed ACE1 alleles of Triticum isolates in the databases and found two new types of alleles, ACE1^Br118.2 and ACE1^T109, in addition to ACE1^Br48 (Fig. 4a and Supplementary Fig. 8). Disruption of ACE1^Br118.2 and ACE1^T109 impaired the aggressiveness of their carriers (Br118.2 and T109) on N4 (Fig. 4b and Supplementary Fig. 9), indicating that they are functional and required for the aggressiveness on wheat. To check interactions between these ACE1 alleles and Pi33, we first chose PO12-7301-2 carrying the nonfunctional ACE1 and a functional SYN2 (Fig. 1c) as a recipient of those alleles. This isolate was virulent on both of rice cultivars, Aichi–Asahi (pi33) and Bala (Pi33) (Fig. 4c), but its transformants carrying ACE1^Br48, ACE1^Br118.2, and ACE1^T109 gained complete avirulence on Bala without losing the virulence on Aichi–Asahi (Fig. 4c). These results suggest that all of these ACE1 alleles function as avirulence genes corresponding to Pi33. On barley, those transformants gained strong aggressiveness as expected. Second, we chose PH297, an MoO isolate carrying nonfunctional ACE1 and SYN2 (Supplementary Fig. 10a), as another recipient. As test rice cultivars, CO39 (pi33) and IR64 (Pi33) were employed because PH297 was avirulent on Bala due to other factor(s). PH297 was virulent on both CO39 and IR64, but its transformants carrying ACE1^Br48, ACE1^Br118.2, and ACE1^T109 again gained complete avirulence on IR64 in spite of lacking a functional SYN2 (Supplementary Fig. 10b). These results suggest that the avirulence on Pi33 is conferred by the ACE1 alleles or the Part A alone, or in other words, that the SYN2 allele is not required for the recognition by Pi33. This is in accordance with a previous report in the MoO - rice interaction that SYN2 is not required for the avirulence on Pi33 rice cultivars²⁶. On barley those transformants did not gain strong aggressiveness, which is reasonable because SYN2 is required for the fungal aggressiveness on wheat, a close relative of barley (Supplementary Fig. 4). Taken together, we suggest that, if Pi33 is transferred from rice to wheat, it may recognize the ACE1 alleles of MoT and function as a gene for resistance to MoT.

Fig. 4: *ACE1* alleles of MoT isolates function as avirulence genes corresponding to rice resistance gene *Pi33.*

A point mutation impairing the ACE1 enzymatic function leads to reduced aggressiveness of MoT on wheat

In the MoO-rice pathosystem, Pi33 has been already defeated by some isolates^25,33 probably through the loss of function of ACE1 as exemplified by PO12-7301-2 and PH297 (Fig. 1c and Supplementary Fig. 10a). A critical concern about the use of Pi33 in wheat breeding is that Pi33 transferred to wheat may also be overcome by new races in future through mutations of the ACE1 alleles in MoT. Böhnert et al.²⁵ suggested that ACE1 enzymatic activity is essential for its avirulence function, based on a fact that a single amino acid exchange (C183A) in the β-ketoacyl synthase domain of ACE1 abolished the recognition of the fungus (MoO) by Pi33. Is the ACE1 enzymatic activity also essential for its function for aggressiveness? To answer this question, we introduced the C183A mutation into ACE1^Br48, fused it to GFP, and established as ace1^BrC183A:GFP. As a control, we also produced ACE1^Br48:GFP by fusing the intact ACE1^Br48 to GFP. These constructs were introduced into PO12-7301-2, and resulting transformants were sprayed on rice cultivars. On Bala (Pi33), transformants with ACE1^Br48:GFP were avirulent but those with ace1^BrC183A:GFP showed virulence with the same level of aggressiveness as PO12-7301-2 (Fig. 5a). On Aichi–Asahi (pi33) those transformants showed the same level of aggressiveness as PO12-7301-2 irrespective of the transgenes (Fig. 5a). On barley ACE1^Br48:GFP conferred the strong aggressiveness on barley to MoO isolate PO12-7301-2, but ace1^BrC183A:GFP did not, suggesting that the C183A mutation impaired the function for conferring strong aggressiveness on barley. In infection assay on wheat, Br48ΔACE1 was used as a recipient of those constructs. The expression of these constructs in appressoria was confirmed by fluorescence microscopy (Supplementary Fig. 11). ACE1^Br48:GFP conferred the strong aggressiveness on wheat cv. N4 to Br48ΔACE1, but the C183A mutation impaired this function (Fig. 5b). These results suggest that P. oryzae can overcome Pi33 through the single amino acid exchange without losing its fundamental aggressiveness on rice, but cannot do so on barley and wheat.

**Fig. 5: A point mutation in a core catalytic residue of *ACE1* abolishes not only the recognition by rice *Pi33* but also the aggressiveness on wheat.**

Discussion

Böhnert et al.²⁵ cloned ACE1 as a gene conditioning the avirulence of MoO isolates on rice cultivars carrying Pi33. It was a unique avirulence gene encoding a hybrid enzyme involved in microbial secondary metabolism²⁵. Collemare et al.²⁶ found that ACE1 is a member of a gene cluster composed of 15 secondary metabolism genes, and designated it as ACE1 cluster. The ACE1 cluster contained SYN2 encoding another PKS-NRPS. These cluster genes displayed the same infection-specific expression pattern during the penetration of the fungus into host (rice) tissues²⁶, suggesting that the gene cluster may play a role in the infection process. Based on the discontinuous distribution of the ACE1 cluster among fungal species, Khaldi et al.³¹ speculated that the metabolite produced by this cluster may be an important pathogenicity factor. However, ACE1 did not appear to contribute to lesion formation, invasive growth²⁵, or aggressiveness on rice cultivars²⁶. SYN2 was not required for the biosynthesis of avirulence signal on Pi33 nor aggressiveness on rice cultivars²⁶. What is the intrinsic role of the ACE1 cluster? The present study showed that the ACE1 cluster plays an important role in aggressiveness of P. oryzae through promoting the successful penetration of cell walls of host plants (Fig. 2). The promotion of penetration was associated with the suppression of formation of fluorescent papillae. This is reasonable from the viewpoint of the timing of ACE1 expression because ACE1 transcription was detected only in mature appressoria, reaching to a maximum 17 h after inoculation³⁴, and then rapidly disappeared once secondary infectious hyphae began to spread²⁵. Interestingly, this contribution of ACE1 to aggressiveness was apparent on barley, wheat, finger millet, and perennial ryegrass, but not detected on rice and foxtail millet (Fig. 3). This may be the reason why Böhnert et al.²⁵ and Collemare et al.²⁶ did not detect the contribution of ACE1 or SYN2 to aggressiveness or pathogenicity on rice. It should be noted that this difference of the contribution to aggressiveness is not attributable to inherent characteristics of the pathotypes or isolates but is dependent on plant species (genera) they attack. For example, the ACE1 cluster in the MoO isolates does not contribute to their aggressiveness on rice (Oryza sativa), but contributes to their aggressiveness on barley (Hordeum vulgare) (Fig. 3). This may be attributable to difference of the sensitivity of each plant species (genus) to product(s) of the ACE1 cluster. The product of the ACE cluster is supposed to be a tyrosine-derived cytochalasin compound³⁵ but is not fully clarified. Further studies are needed to reveal how the product of the ACE1 cluster promotes the successful penetration of cell walls or is involved in the suppression of papilla formation.

Most of newly introduced disease resistance genes in crops have been rendered ineffective by emergence of new pathogen races which escaped the recognition by the resistance genes through the loss of function or modification of their corresponding avirulence genes³⁶. However, if an avirulence gene is indispensable for the survival of the pathogen and unable to accommodate itself so as to escape the recognition without losing its function for survival, the resistance gene should be durable^17,18. We found that the ACE1 cluster of MoT was indispensable for its strong aggressiveness on wheat, but also contained alleles of avirulence gene ACE1 recognized by rice resistance gene Pi33 (Fig. 4). Furthermore, a point mutation of the ACE1 alleles in MoT for evading the recognition by Pi33 inevitably caused critical reduction of its function for aggressiveness on wheat (Fig. 5). It seems impossible for MoT to evade the recognition by Pi33 without losing its aggressiveness on wheat because both functions of ACE1 (for avirulence and aggressiveness) are considered to be dependent on its enzymatic activity²⁵ (Fig. 5). From these results, we suggest that Pi33 may be durable if transferred from rice to wheat, or in other words, that Pi33 may be used as an arrow targeting an Achilles heel of MoT.

Considering that resistance genes effective against MoT are rarely found in the wheat population, it is a promising strategy to look for such genes in rice which has a long history of interactions with MoO. Navia-Urrutia et al.³² found that a homolog of AvrPiz-t in MoT was recognized by Piz-t in rice. Then, they produced transgenic wheat plants expressing the rice Piz-t gene and inoculated them with a MoT isolate carrying the functional AvrPiz-t homolog. Although the transformants did not express a useful level of resistance in spikes, some of them showed a significant reduction of disease progress at the seedling stage. This result suggests that blast resistance genes in rice can be expressed and used in wheat. Since this strategy (transfer of rice genes to wheat) involves transgenic methods, the deficits such as low levels of expression or low levels of resistance in spikes will be overcome by manipulation of the transgene. Pi33 has been mapped on the short arm of rice chromosome 8³³ and narrowed down to its 240 kb region³⁷, but not yet isolated. The cloning of Pi33 is awaited for producing transgenic wheat plants carrying it and testing their reactions to wheat blast.

It should be noted that Pi33 in rice has been already defeated by some MoO isolates^25,33 as exemplified by PO12-7301-2 (Figs. 1, 4) and PH297 (Supplementary Fig. 10). This difference of predicted durability of Pi33 in rice and wheat is attributable to the difference of contribution of ACE1 alleles to aggressiveness on these crop species (Figs. 1e, 3b). The results shown in Fig. 3 further suggest that Pi33 may also be used for breeding of barley, finger millet, and ryegrass as a durable resistance gene. Resistance genes that have been already defeated in one crop species may be revived as a durable resistance gene in other crop species.

Methods

Fungal materials and genetic crosses of P. oryzae

Pyricularia oryzae isolates used were PO12-7301-2 (MoO), Ken53-33 (MoO), PH297　(MoO), IN77-20-1-1 (MoS), MZ5-1-6 (MoE), TP2 (MoL), Br48 (MoT), Br118.2 (MoT), and T109 (MoT) (Supplementary Table 2). PH297 and T109 (original name, BTGP-6(e)) were provided by Dr. C. J. R. Cumagun, University of the Philippines, Los Banõs, the Philippines, and Dr. Md Tofazzal Islam, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh, respectively. In addition to these wild isolates, 54N1, an F₁ culture derived from a cross between PO12-7301-2 and Br48²³, was used to generate a BC₁F₁ population for linkage mapping of Pwt2. 54N1 was crossed with Br48 on oatmeal agar media as described previously²². One ascospore was isolated from each ascus so that each of the resulting hybrids was derived from an independent meiotic event.

Plant materials

The plant materials used were Triticum aestivum cv. Norin 4 (N4), Hordeum vulgare cvs. H.E.S.4 and Nigrate, Setaria italica cv. Aka-awa (Si5), Eleusine coracana cv. Purna (Ec19), Lolium perenne cv. Friend, and Oryza sativa cvs. Aichi–Asahi, CO39, Bala, and IR64. Bala and IR64 were provided by the International Rice Research Institute (IRRI), the Philippines, and Dr. R. Ishikawa, Kobe University, Japan, respectively.

Infection assay

Wheat and barley seeds were sown in vermiculite supplied with liquid fertilizer in a seedling case (5.5 × 15 × 10 cm), and grown at 22 °C in a controlled environment room with a 12 h photoperiod of fluorescent lighting for 8 days. Foxtail millet and finger millet were sown in Prime Mix soil (Sakata seed corporation, Yokohama, Japan) in the seedling case, and grown at 26 °C in a growth chamber with a 12 h photoperiod of fluorescent lighting for 2 weeks. Perennial ryegrass seeds were sown in Prime Mix soil in the seedling case, and grown at 22 °C in a phytotron chamber with a 12 h photoperiod of artificial lighting with metal halide lamps for 3 weeks. Rice seeds were sown in Ube soil (Zen-noh, Tokyo, Japan) in the seedling case, and grown at 26 °C in a natural-light cabinet for 3 weeks. Inocula were prepared as described previously³⁸. Conidial suspension (1.0 × 10⁵ conidia per mL) with 0.01% Tween 20 was sprayed on primary (wheat and barley), third (foxtail millet and perennial ryegrass), and fourth (finger millet and rice) leaves fixed onto a hard plastic board with an air compressor. The cases were sealed to maintain high humidity and placed in darkness for 24 h at 22 °C or 26 °C. The inoculated seedlings were then transferred to a growth chamber with a 12 h photoperiod of fluorescent lighting and incubated further at the same temperature as in darkness. Four to five days after inoculation, symptoms were evaluated based on the lesion areas formed by fungal infection. For each leaf, the leaf size and lesion area were measured using ImageJ (https://imagej.nih.gov/ij/), and the relative area covered by lesions (percentage of lesion area) was calculated.

Construction of a BAC library

Genomic DNA of Br48 was digested with Sau3AI, and ~100 kb fragments were ligated with pCC1BAC/BamHI vector (EPICENTRE). The ligation products were transformed into E. coli ElectroMAX DH10B (Invitrogen) through electroporation.

Construction of plasmids

Details of the plasmid construction and the primer sequences used are described in Supplementary Table 3. Their outlines are briefly described below. To construct an ACE1^Br48 complementation vector, a ~ 15 kb EcoRI fragment containing ~1.6 kb upstream, ORF, and ~1.4 kb downstream sequences of ACE1^Br48 was liberated from ZT1-1-A (a BAC clone of the Br48 genomic library), cloned into pBlueScript II SK(+) and established as pACE1^Br48. Other ACE1/SYN2 complementation vectors were constructed by assembling genomic fragment(s) containing ~1.6–1.7 kb upstream, ORF, and ~1.4 kb downstream sequences and a vector backbone of pBlueScript II SK(+) via In-Fusion cloning (Takara Bio, Otsu, Japan). These genomic fragments were either PCR-amplified from genomic DNA of P. oryzae isolates or liberated from a BAC clone of Br48.

To generate ACE1-KO (knock out) mutants, a KO vector was constructed by replacing a ~ 3.6-kb AgeI fragment of pACE1^Br48 with a hygromycin resistance gene cassette. The hygromycin resistance gene cassette was PCR-amplified from pSH75³⁹ with primers HygR_F2 and HygR_R2. To generate SYN2- and ACE1 cluster-KO mutants, KO vectors were constructed by assembling ~2.5-3.1-kb upstream and downstream fragments of the target gene/region, a hygromycin resistance gene cassette, and a vector backbone of pBlueScript II SK(+) via In-Fusion cloning. To construct CRISPR/Cas9 expression vectors targeting genes/regions of interest, sense, and antisense oligonucleotides were annealed and inserted to pCRISPR/Cas-U6-1⁴⁰ by Golden Gate cloning as described in Arazoe et al.⁴⁰.

To monitor the expression of ACE1 proteins, we constructed a vector encoding a C-terminal fusion of ACE1 with GFP and expressed under the control of the ACE1 promoter and terminator sequences. A 16.4 kb NotI fragment containing ~1.6 kb upstream and N-terminal part of the coding sequences of ACE1^Br48 and the vector backbone of pBlueScript II SK(+) was liberated from pACE1^Br48. The C-terminal part of the ACE1^Br48 coding sequence lacking the stop codon was PCR-amplified from pACE1^Br48 with primers pACE1_N_F and ACE1woStop_R. A ~ 0.7 kb GFP gene fragment was PCR-amplified from pEGFP75⁴¹ with primers GFP_i_F and GFP_i_R. A ~ 1.4-kb downstream sequence of ACE1^Br48 was PCR-amplified from pACE1^Br48 with primers ACE1ter_F and pACE1_N_R. The four fragments were assembled by InFusion reaction to establish pACE1^Br48-GFP.

To introduce C183A mutation in ACE1^Br48-GFP, a DNA fragment carrying “TGC” to “GCT” mutation in the codon position for the 183rd amino acid of ACE1^Br48 was generated by annealing two oligos, F_C183A and R_C183A. Upstream and downstream flanking sequences of the mutation target site were PCR-amplified from pACE1^Br48-GFP with primers ACE1_m_F1 and ACE1_m_R1 and ACE1_m_F2 and ACE1_m_R2, respectively. To replace the corresponding sequence in pACE1^Br48-GFP, the three DNA fragments were integrated via InFusion reaction with an 18.6 kb BspEI/FseI double-digestion fragment liberated from pACE1^Br48-GFP. The resultant plasmid was established as pACE1^BrC183A-GFP.

Transformation of P. oryzae

Protoplasts of P. oryzae were prepared and transformed as described previously⁴². In the complementation tests, vectors carrying genes from the ACE1 cluster were introduced into P. oryzae isolates/strains through co-transformation with pSH75 containing the hph gene for hygromycin-B selection or pII99⁴³ containing the nptII gene for geneticin selection. In targeted gene disruption, KO vectors were introduced alone or together with CRISPR/Cas9 expression vectors into protoplasts. Transformants carrying transgenes were selected by colony PCR. Details of the transformants produced in this study are described in Supplementary Table 4.

Microscopy

For microscopic observation of infection behaviors of ACE1-KO mutants, primary leaves of wheat cv. N4 were sampled at 48 h after inoculation and boiled in alcoholic lactophenol (lactic acid/phenol/glycerol/distilled water/ethanol = 1:1:1:1:8, vol/vol/vol/vol/vol) for 2 min for fixation. Cytological response of the specimens was observed under bright and dark fields of a fluorescence microscope (Olympus, Tokyo, Japan) with an exciter filter B. Fluorescence of the ACE1-GFP fusion protein was observed using the fluorescence microscope at 20 h after inoculation of glass coverslips with conidial suspensions.

Detection of ACE1 cluster genes and phylogenetic analysis

Protein sequences of 15 genes within the ACE1 cluster (Fig. 3) were retrieved from the genome annotation of P. oryzae strain 70-15 (version MG8). The protein sequences were aligned to genome assemblies of P. oryzae isolates listed in Supplementary Tables 2 and 5 via exonerate⁴⁴ with “-m p2g --percent 50 -n 1” options. Homologous sequences in each isolate and the identity to the query protein sequence were retrieved from the exonerate output. Protein sequences of the ACE1 alleles from different isolates were aligned by MUSCLE⁴⁵ in MEGA version 11⁴⁶. A maximum likelihood tree was inferred by IQtree version 2.2.0.3⁴⁷ and branch supports were obtained with the ultrafast bootstrap⁴⁸. The phylogenetic tree was visualized by using iTOL⁴⁹.

Statistics and reproducibility

For the assessment of fungal virulence, data from 17-45 biologically independent samples from three independent experiments were collected per strain, except for Supplementary Figs. 7b (13-16 biologically independent samples from two independent experiments per strain) and 9a (9-17 biologically independent samples from a single experiment per strain). For the assessment of the host invasion behavior, data from 9 biologically independent samples from three independent experiments were collected per strain. Significant differences between groups were determined using the Dunn’s test (Figs. 1d, e, f, and 5b, and Supplementary Figs. 3a, b, 4b, c, 7a, b, c, d, 9a, and b) or Tukey test (Fig. 2a, b, and c) at 5% level.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Nucleotide sequence data reported are available in the DDBJ Sequenced Read Archive under the accession number PRJDB14584. Source data and original gel images are available in Supplementary Data 1 and Supplementary Fig. 12, respectively. Any additional data supporting the findings of this study are available from the corresponding author upon reasonable request. Fungal genome sequence data used in this study are listed in Supplementary Table 5. All plasmids, plant lines, and fungal strains employed or generated in this work are available from the authors upon request.

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Acknowledgements

We thank Dr. Md Tofazzal Islam, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh, and Dr. C. J. R Cumagun, a former professor of the University of the Philippines, Los Banõs, for providing the wheat blast isolates collected in Bangladesh and the rice blast isolates collected in the Philippines, respectively. We also thank the International Rice Research Institute (IRRI), the Philippines, and Dr. R. Ishikawa, Kobe University, Japan, for providing the rice cultivars carrying Pi33, and Dr. K. Ikeda, Kobe University, for technical assistance in microscopic observations. Special thanks are due to Dr. R. Terauchi, Kyoto University, for giving valuable suggestions. This research was supported by JSPS KAKENHI Grant Numbers, JP17H01462 and JP21H04726, and the Research Program on Development of Innovative Technology grants (JPJ007097) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN).

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These authors contributed equally: Trinh T. P. Vy, Yoshihiro Inoue.

Authors and Affiliations

Graduate School of Agricultural Science, Kobe University, Kobe, 657-8501, Japan
Trinh T. P. Vy, Soichiro Asuke, Hitoshi Nakayashiki & Yukio Tosa
Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan
Yoshihiro Inoue
Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan
Izumi Chuma

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Trinh T. P. Vy
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Yoshihiro Inoue
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Y.T., Y.I., and T.T.P.V. designed research; S.A., I.C., and H.N. provided methodology and some data; T.T.P.V. performed research; T.T.P.V., Y.I., and Y.T. analyzed data; Y.I. and Y.T. wrote the paper.

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Correspondence to Yukio Tosa.

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Vy, T.T.P., Inoue, Y., Asuke, S. et al. The ACE1 secondary metabolite gene cluster is a pathogenicity factor of wheat blast fungus. Commun Biol 7, 812 (2024). https://doi.org/10.1038/s42003-024-06517-7

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Received: 04 August 2023
Accepted: 27 June 2024
Published: 04 July 2024
DOI: https://doi.org/10.1038/s42003-024-06517-7

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