Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in

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Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in Pyricularia oryzae Choong-Soo YUN, Takayuki Motoyama, and Hiroyuki Osada ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00353 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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1

Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in Pyricularia oryzae

2

Choong-Soo Yun, Takayuki Motoyama, and Hiroyuki Osada*

3 4

Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, 2-1

5

Hirosawa, Wako-shi, Saitama 351-0198, Japan

6 7

*Correspondence: Tel: +81-48-467-9541; Fax: +81-48-462-4669; Email: [email protected]

8 9 10 11 12

ABSTRACT

13

Tenuazonic acid (TeA) is a mycotoxin produced by the rice blast fungus Pyricularia oryzae

14

and some plant pathogenic fungi. We previously demonstrated that TeA is biosynthesized in P.

15

oryzae by TeA synthetase 1 (TAS1), and that its production is induced by osmo-sensory

16

MAPK-encoding gene (OSM1) deletion or the addition of 1% DMSO to cultures; however, the

17

regulatory mechanisms of TeA production were unknown. Here, we identified a

18

Zn(II)2-Cys6-type transcription factor in the upstream region of TAS1, which is encoded by TAS2

19

and regulates TeA production. We also found PoLAE1, which is a homolog of LaeA, a regulator

20

of fungal secondary metabolism. Analysis of PoLAE1 deletion and overexpression strains

21

indicated that PoLAE1 drives TeA production. We also demonstrated that 2 TeA-inducing

22

signals, 1% DMSO addition and OSM1 deletion, were transmitted through PoLAE1. Our results

23

indicate that TeA production is regulated by 2 specific regulators, TAS2 and PoLAE1, in P.

24

oryzae.

25

Word count: 149 1



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Filamentous fungi are well-known producers of a variety of secondary metabolites (SMs),

27

including mycotoxins, antibiotics, and other pharmaceutically useful small molecules. Recent

28

genome sequencing has revealed that filamentous fungi genomes contain more SM

29

biosynthesis-related genes than once believed. However, many of these genes remained inactive

30

under general laboratory culture conditions (1). Transcription of these SM biosynthesis-related

31

genes is precisely and complexly regulated in fungi in response to various environmental signals.

32

Elucidation of the regulatory mechanism of these SM biosynthesis genes is important for a

33

better understanding of the interaction between fungi and their environments. In particular,

34

understanding the regulatory mechanism of mycotoxin biosynthesis is essential for the control

35

of mycotoxin production to protect human and animal health.

36

In general, SMs are biosynthesized in fungi via the cooperation of clustered genes; many

37

include a gene for a cluster-specific DNA binding binuclear Zn(II)2Cys6-type transcription factor,

38

a type known to be unique to fungi, which activates the transcription of the clustered genes to

39

produce an SM (2). Examples of the transcription factor genes include aflR for aflatoxin

40

biosynthesis in Aspergillus nidulans (3), gliZ for gliotoxin biosynthesis in A. fumigatus (4), tri6

41

for trichothecene biosynthesis in Fusarium sporotrichioides (5), and ctnA for citrinin

42

biosynthesis in Monascus purpureus (6).

43

SM production in fungi is also regulated by upper-level regulators than cluster-specific

44

transcription factors. These upper-level, called global, regulators are trans-acting positive or

45

negative transcriptional regulators of SM gene clusters. LaeA (Loss of aflR expression) is a

46

well-known global regulator of secondary metabolism in Aspergillus spp. Disruption of LaeA

47

caused decreased production of penicillin, terrequinone A, and sterigmatocystin by A. nidulans

48

and gliotoxin by A. fumigatus (7). Genome-wide transcriptional analysis of the wild-type and

49

LaeA mutant strains also indicated that approximately 40% of key SM genes were under the

50

regulation of LaeA in A. fumigatus (8). Although its functions have not yet been fully 2


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characterized, LaeA has sequence similarity to histone and arginine methyltransferases, and is

52

assumed to control chromatin remodeling to regulate the transcription of SM gene clusters (9).

53

It is also reported that LaeA composes the heterotrimeric velvet complex with VelB and VeA to

54

control the secondary metabolism in A. nidulans (10). LaeA orthologs have also been identified

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in other fungi, and are reported to be involved in production control of SMs including

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beauvericin, ferricrocin, fusaric acid, and trichothecenes in Fusarium spp., T-toxin and melanin

57

in Cochliobolus heterostrophus, and monacolin K in M. pilosus (11).

58

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic

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fungi like Alternaria spp., Epicoccum sorghinum, and Pyricularia oryzae (synonym,

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Magnaporthe oryzae). TeA has been detected in a wide variety of TeA-producing

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fungi-contaminated crops, fruits, and vegetables (12, 13). TeA is the most toxic of the Alternaria

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toxins. It inhibits protein biosynthesis on ribosomes by suppressing the release of new proteins

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(14). TeA has acute toxicity in mammals, with an oral median lethal dose of 182 or 225 mg kg-1

64

body weight and 81 mg kg-1 body weight for male and female mice, respectively (15, 16).

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Accordingly, the European Food Safety Authority evaluated the toxicological potential of TeA

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and determined its threshold of toxicological concern to be 1,500 ng kg-1 body weight d-1 (17).

67

Recently, we identified the biosynthetic gene for TeA in P. oryzae by first determining 2

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TeA-inducing conditions: osmo-sensory mitogen activate protein kinase (MAPK) -encoding

69

gene (OSM1) deletion and 1% dimethyl sulfoxide (DMSO) in the culture conditions (18); in this

70

paper, we describe the regulatory mechanism of TeA production in P. oryzae.

71

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RESULTS AND DISCUSSION

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Zn(II)2-Cys6-type cluster transcription factors specific for fungal SM gene clusters are usually

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found within the clusters; they positively co-regulate clustered gene transcription to produce

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specific SMs. Unlike the other fungal SMs, only 1 enzyme (TeA synthetase 1, TAS1) is fully

76

sufficient to produce TeA, suggesting that there is no clustered gene need to regulate

77

simultaneously. However, in an analysis of the open reading frames (ORFs) that exist in the

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region surrounding TAS1, we found a putative Zn(II)2-Cys6-type transcription factor encoding

79

gene MGG_07800 (Figure 1a). To verify that this gene is involved in the production of TeA, we

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constructed MGG_07800 knockout strains (Figure 1b, c), and analyzed their metabolites with

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ultra-performance liquid chromatography–mass spectroscopy (UPLC–MS). The knockout strain

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lost the capability to produce TeA under the DMSO-added, TeA-inducing culture condition

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(Figure 1d), indicating that the gene product is a positive transcription factor for TAS1. Thus, we

84

renamed MGG_07800 as TAS2. We also confirmed by quantitative polymerase chain reaction

85

(PCR) that TAS2 is required for induced expression of TAS1 (Supplementary Figure 1a).

86

Alternaria spp. and E. sorghinum are known as famous TeA producers, and their genome

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sequences have already been reported (19, 20). To find TAS2 homologs in their genomes, we

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investigated the surrounding genome regions of TAS1 homologs. Interestingly, neither of them

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have the TAS2 homolog. Among the Basic Local Alignment Search Tool (BLAST) search

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results of TAS1 and TAS2, only one fungus besides P. oryzae (Thermothelomyces thermophile,

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which is phylogenetically closely related to P. oryzae) has both TAS1 and TAS2 homologs

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(XP_003660956.1 and XP_003660954.1). This means that the TeA production regulatory

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mechanism of Alternaria spp. and E. sorghinum is different from that of P. oryzae. Identical SM

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production via divergent regulatory mechanisms in different fungi is a very unique case.

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The DNA binding domains in Zn(II)2-Cys6-type transcription factors interact with similar DNA

sequences.

AflR

from

Aspergillus

spp.

is

the

4


Zn(II)2-Cys6-type

aflatoxin

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biosynthesis-specific regulator; its DNA binding sequences in the promoter regions of target

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genes have been intensively studied, and the consensus binding sequence 5′-TCG(N5)CGR-3′

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(R = G or A) in the promoter region of a target gene was identified as an AflR binding site (21).

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TAS1 also has a 5′-TCG(N5)CGG-3′ binding sequence in the promoter region (position –140 to

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–150 from the TAS1 start codon) and TAS2 may be able to bind this sequence to regulate TAS1

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expression.

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The first reported LaeA ortholog outside the class of Eurotiomycetidae, which includes

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Aspergillus spp., is FfLae1 from F. fujikuroi of the class Sordariomyceta (22). P. oryzae also

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belongs to Sordariomyceta; therefore, we searched for proteins with sequence similarity to

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FfLae1 in the P. oryzae genome sequence using the BLAST. In the BLAST search results, we

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found 1 LaeA homolog candidate gene (MGG_01233) that shares 34% amino acid identity with

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FfLae1 and has a methyltransferase domain, like the other reported LaeA orthologs. To

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investigate whether MGG_01233 is involved in TeA production in P. oryzae, we constructed

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MGG_01233 disruptants (Figure 2a, b). The constructed disruptants lost the capability to

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produce TeA under the DMSO-added, TeA-inducing culture condition (Figure 2e). We also

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constructed MGG_01233-overexpressing strains by inserting the TEF1 promoter from A. oryzae

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upstream of the MGG_01233 start codon via homologous recombination (Figure 2c, d). UPLC–

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MS analysis of the extracted metabolites from the resulting strains showed TeA-producing

115

capability without DMSO addition (Figure 2e). The transcriptional levels of TAS1 in

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MGG_01233 mutants coincided with the TeA production levels (Supplementary Figure. 1a). We

117

renamed MGG_01233 to PoLAE1. These results suggest that TeA production is positively

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controlled by PoLAE1 in P. oryzae.

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In this study, we found 2 positive transcriptional regulators, TAS2 and PoLAE1, for TeA

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production. However, the positional relationship of these 2 transcription factors was still unclear.

121

To investigate the positional relationship between TAS2 and PoLAE1, we constructed 5



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PoLAE1-overexpressing TAS2 deletion strains (Figure 3a). The constructed strains did not

123

produce TeA, although the PoLAE1 overexpression strains produce TeA. This observation

124

suggested that TAS2 regulates TeA production downstream of PoLAE1 (Figure 3d).

125

It is reported that global regulators, LaeA and its orthologs, regulate production of a number

126

of SMs in one fungus. However, in P. oryzae, PoLAE1 deletion or overexpression strains did not

127

show notable changes in production of SMs other than TeA (Supplementary Figure. 2).

128

Additionally, growth rate and conidiation were not affected in TAS2 or PoLAE1 mutants.

129

P. oryzae Kita1 is a low-level TeA-producing strain. In our previous work, we identified 2

130

TeA-inducing conditions. One is the addition of 1% DMSO to the culture medium and the other

131

is the deletion of the OSM1, which functions downstream of the two-component signal

132

transduction system. We found that 1% DMSO did not induce TeA production in the PoLAE1

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deletion strains (Figure 2e), indicating that the DMSO-induced signal is transmitted through

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PoLAE1. Next, we investigated the role of OSM1 in the induction of TeA. To investigate if

135

OSM1 deletion induced TeA production via PoLAE1, we constructed PoLAE1 disruption strains

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in an OSM1 deletion strain (Figure 3b, c). The constructed OSM1 and PoLAE1 double deletion

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mutants lost the capability to produce TeA, indicating that the TeA-inducing signal generated by

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OSM1 deletion is transmitted via PoLAE1 (Figure 3d). We also analyzed the expression of

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LAE1, TAS2, and OSM1 in the different genetic backgrounds and conditions (Supplementary

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Figure 1b, c, d). However, expression of these genes was not well correlated with TeA

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production, suggesting that transcriptional control of these genes is not important.

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In filamentous fungi, MAPK has been reported to be involved in the regulation of SMs and

143

responses to environmental stress signals. The F. graminearum MAPK-encoding gene FgOs2

144

deletion strain showed enhanced pigmentation and failed to produce the mycotoxin

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trichothecene (23). The A. nidulans MAPK-encoding gene mpkB deletion mutant also showed

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reduced mycotoxin production levels. Furthermore, due to lower expression levels of LaeA in 6


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the mpkB deletion strain, the reduced SM production after mpkB deletion was assumed to be

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caused via LaeA (24). Our data from the P. oryzae OSM1–PoLAE1 double deletion mutant

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clearly showed that increased SM production caused by MAPK interference is mediated by

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PoLAE1. This is the first direct evidence that the p38 MAPK signal is transmitted through

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LAE1 to regulate secondary metabolite production. (Figure 3d, Supplementary Figure. 1a).

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DMSO is a highly polar solvent that dissolves both polar and nonpolar chemical compounds,

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and is frequently used as a solvent for chemical compounds in biological activity studies. In the

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course of screening for secondary metabolism-inducing chemical compounds, we found that 1%

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DMSO induced the production of TeA in P. oryzae. As the chemical compounds were dissolved

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in DMSO, all tested compounds induced TeA production. Enhanced production of bacterial SMs

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upon the addition of DMSO to cultures has also been reported. With the addition of 3% DMSO,

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chloramphenicol and tetracenomycin C production increased approximately 3-fold in

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Streptomyces venezuelae and S. glaucescens, respectively (25). However, their precise

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mechanisms of induction were unknown.

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DMSO inhibits the growth of some species of fungi. Candida spp. showed marked growth

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inhibition in 2% DMSO, while 1% or lower concentrations caused no notable effects (26). The

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growth inhibition by 2% DMSO in P. oryzae cultures was substantial; however, 1% DMSO

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addition did not inhibit growth, and 0.2% DMSO addition did not induce TeA production.

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Growth inhibition indicates that fungi are under stress, and 1% DMSO may stress P. oryzae,

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although growth looks normal. Thus, we analyzed whether general stress is related to induced

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production of TeA or not. Histone modification is known to be altered in numerous

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stress-related conditions. Thus, we have treated P. oryzae with the histone deacetylase inhibitor

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suberoylanilide hydroxamic acid (SAHA) and the DNA methyltransferase inhibitor

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5-azacytidine (Aza) with a dosage of 20 to 2,000 µΜ. Neither SAHA nor Aza could induce

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production of TeA in P. oryzae, in spite of the presence of severe growth inhibition conditions 7



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(2,000 µΜ Aza treatment) (Supplementary Fig. 3). This means that growth inhibition stress is

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not the reason for induced production of TeA by DMSO addition. At this time, the mechanism

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of TeA production induction by DMSO addition is uncertain, and further study will be required

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to clarify it.

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In summary, we found 2 transcription factors, TAS2 and PoLAE1, which positively regulate

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TeA production in P. oryzae. PoLAE1 regulated TeA production via TAS2. We also determined

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that 2 TeA-inducing signals, 1% DMSO addition and OSM1 deletion, are transmitted through

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PoLAE1. Mycotoxin biosynthesis is associated with a complex biological network involved in

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diverse aspects of cellular function. We expect that the results of this study will aid further

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elucidation of TeA regulatory mechanisms.

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METHODS

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Strains and culture conditions. The strains and plasmids used in this study are listed in

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Supplementary Table 1. The rice plant pathogenic P. oryzae strain Kita1 was used as a

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wild-type strain. P. oryzae was grown on PDA plates (3.9% potato dextrose agar; Difco) or

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OMA plates (5% oatmeal agar; Difco) at 25 °C. For SM analysis, P. oryzae was grown in 100

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µl of liquid YG media (0.5% yeast extract and 2% glucose) in a 96 well flat bottom plate

189

(Iwaki) at 25 °C without agitation for 5 d (static-culture). P. oryzae was transformed as

190

described previously (27) using the Agrobacterium tumefaciens-mediated transformation

191

(ATMT) method (28). P. oryzae transformants were selected with 500 µg mL-1 hygromycin B

192

or 150 µg mL-1 blasticidin S. Escherichia coli DH5α were grown in Lysogeny broth (LB) at 37

193

°C, and transformation was performed with a standard method (29). We added 50 µg mL-1

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kanamycin (Km) to the E. coli-transformant selective medium.

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Gene disruption. The construction of TAS2 (MGG_07800) or PoLAE1 (MGG_01233)

196

disruptants was performed by exchanging a whole-gene ORF with the hygromycin B or

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blasticidin S resistance gene expression unit via a homologous recombination-based gene

198

replacement system. PoLAE1 gene disruptants were constructed as follows. Two kb of the

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upstream and downstream regions of PoLAE1 was amplified from the genomic DNA of P.

200

oryzae via PCR with the primers ∆PoLae1_UP-F and ∆PoLae1_UP-R for the upstream region

201

(fragment 1), and the primers ∆PoLae1_DN-F and ∆PoLae1_DN-R for the downstream region

202

(fragment 2). The primers 5HPH and 3HPH were used to amplify the hygromycin B or

203

blasticidin S resistance gene expression units from pBI_M07803::HPH or pBI_OE::TAS1 (18)

204

(fragment 3). Simultaneously, the vector sequence of pBI121 (Clontech) between the right and

205

left borders was amplified via PCR with the primers pBI121-RB and pBI121-LB (fragment 4).

206

The fragments were gel-purified and cloned using the In-Fusion® cloning technology to yield

207

pBI-PoLAE1::HPH or pBI-PoLAE1::BS. The In-Fusion® reaction mixtures were used to 9



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208

transform E. coli DH5α, and transformants were selected with Km (50 µg mL-1). After

209

verification of the inserted DNA sequence, plasmid DNA containing the correct insert was

210

transformed into A. tumefaciens. PoLAE1 disruptants were selected via PCR with the primers

211

∆PoLAE1_CHK_F and ∆PoLAE1_CHK_R, which hybridize outside PoLAE1. This primer set

212

can amplify 1.5 kb of the PoLAE1 ORF from wild-type strains, or the 1.6 kb hygromycin B or

213

1.2 kb blasticidin S resistance gene expression unit from PoLAE1 disruptants. MGG_07800

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disruptants were constructed as described above. Briefly, the primers ∆M07800_UP-F and

215

∆M07800_UP-R for the upstream region (fragment 1), and the primers ∆M07800_DN-F and

216

∆M07800_DN-R for the downstream region (fragment 2) were used for fragment amplification.

217

Fragments 3 and 4 were amplified using the same primers as above. MGG_07800 disruptants

218

were selected via PCR with the primers ∆M07800_CHK_F and ∆M07800_CHK_R, which

219

hybridize outside MGG_07800. This primer set can amplify 2.6 kb of the MGG_07800 ORF

220

including region from wild-type strains, and the 1.8 kb hygromycin B resistance gene

221

expression unit including region from the MGG_07800 disruptants. The primers used are listed

222

in Supplementary Table 1.

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REFERENCES

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mycotoxin

tenuazonic

acid

in

cereals

by

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chromatography-electrospray ionization ion-trap multistage mass spectrometry after 11


high-performance

liquid


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FIGURE LEGENDS

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Figure 1 Identification of a transcription factor that controls TeA biosynthesis. a) Schematic

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representation of the gene location in the TeA biosynthetic gene (TAS1/MGG_07803) flanking

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region. b) Scheme of MGG_07800 gene disruption. Arrows indicate the positions of primers

312

used for disruptant selection. c) PCR analysis of P. oryzae Kita1 (wild-type (WT)) and

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disruptants. The WT fragment was amplified from genomic DNA, and the disrupted fragment

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was amplified with colony PCR. d) UPLC analysis of metabolites extracted from WT,

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∆MGG_07800, and their DMSO-added cultures. Each strain was static-cultured for 5 d at 25 °C.

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A TeA standard (TeA, 100 µM) was also analyzed.

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Figure 2 Relationship between the global transcriptional regulator PoLAE1 and TeA

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biosynthesis. a) Scheme of PoLAE1 gene disruption. Arrows indicate the positions of primers

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used for disruptant selection. b) PCR analysis of P. oryzae Kita1 (WT) and disruptants. The WT

321

fragment was amplified from genomic DNA, and the disrupted fragment was amplified with

322

colony PCR. c) Scheme of PoLAE1 gene overexpression. Arrows indicate the positions of

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primers used for selection of the overexpression strains. d) PCR analysis of P. oryzae Kita1

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(WT) and mutants. The WT fragment was amplified from genomic DNA, and the

325

overexpression strain fragment was amplified with colony PCR. e) UPLC analysis of

326

metabolites extracted from cultures of the ∆PoLAE1, DMSO-added ∆PoLAE1, and PoLAE1

327

overexpression strains. Each strain was static-cultured for 5 d at 25 °C.

328 329

Figure 3 Analysis of the TeA biosynthesis regulation network. a) PCR analysis of WT and

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PoLAE1 overexpression–MGG_07800 disruption strains. The WT fragment was amplified from

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genomic DNA, and the fragment from the double mutants was amplified with colony PCR. b)

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Scheme of PoLAE1 gene disruption of an OSM1 disruption strain. Arrows indicate the positions 14


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of primers used for disruptant selection. c) PCR analysis of WT and double disruption mutants

334

of PoLAE1–OSM1. The WT fragment was amplified from genomic DNA, and the fragment

335

from the double disruption mutants was amplified with colony PCR. The primers used to

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confirm OSM1 disruption are described in previous work and the amplified fragment was

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digested with EcoRV (18). d) UPLC analysis of metabolites extracted from cultures of an OSM1

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disruption strain, an OSM1–PoLAE1 double disruption strain, a DMSO-added OSM1–PoLAE1

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double disruption strain, and a PoLAE1 overexpression–MGG_07800 disruption strain. Each

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strain was static-cultured for 5 d at 25 °C.

341 342

Supporting Information Available: Supplementary Methods (DNA manipulation, Gene

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overexpression, Metabolite extraction and analysis, and Quantitative PCR), supplementary

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Table 1, supplementary Figure 1, 2, and 3. This material is available free of charge via the

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internet at http://pubs.acs.org.

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Regulatory Mechanism of Mycotoxin Tenuazonic Acid Production in

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