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Article

Functional Analysis of MoMyb13, a Myb Transcription Factor Involved in Regulating Growth, Conidiation, Hydrophobicity, and Pathogenicity of Magnaporthe oryzae

by
Ya Li
1,2,
Xiuxia Zheng
1,
Mengtian Pei
1,
Mengting Chen
1,
Shengnan Zhang
1,
Chenyu Liang
1,
Luyao Gao
1,
Pin Huang
1 and
Stefan Olsson
1,3,*
1
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
Fujian Universities Key Laboratory for Plant-Microbe Interaction, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
Synthetic Biology Center, College of Future Technologies, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(2), 251; https://doi.org/10.3390/agronomy14020251
Submission received: 30 December 2023 / Revised: 20 January 2024 / Accepted: 23 January 2024 / Published: 24 January 2024
(This article belongs to the Section Pest and Disease Management)
Browse Figures
Versions Notes

Abstract

:
The Myb family of transcription factors (TFs) is a large and functionally diverse group found in all eukaryotes. Its role in fungi remains poorly studied, despite the fact that it is thought to play a role in the pathogenicity of fungal pathogens. In this study, we have characterized the functional role of a Myb family TF called MoMyb13 in the rice blast fungus, Magnaporthe oryzae. MoMyb13 has orthologues only in ascomycete fungi, making it of special interest. Localization experiments confirmed that MoMyb13 is located in the nuclei, as expected for a TF. Phenotypic analysis showed that MoMyb13 mutants exhibited reduced growth, white instead of dark colonies, formed no conidia and, consequently, no conidial appressoria. The mutants completely lost pathogenicity, despite being able to form dark hyphal appressoria at their hyphae ends. Furthermore, the mutant colonies lost hydrophobicity and had significantly reduced expression of the hydrophobin MPG1 that MoMyb13 appears to regulate. However, overexpression of MPG1 in the mutants restored hydrophobicity, but not pathogenicity. Stress assay showed that the mutants were more sensitive to SDS, CR, and H2O2, but more tolerant to NaCl and SOR. In summary, our study revealed the crucial function of MoMyb13 in the growth, conidiation, hydrophobicity, stress response, and pathogenicity of M. oryzae. MoMyb13 is thus needed in the late and very early stages of infection for the spreading of the fungus to other plants and the early establishment of infection in other plants.

1. Introduction

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a highly destructive disease that affects rice production around the world. It is estimated that this disease causes economic damage worth approximately $66 billion annually, which is enough to feed 60 million people [1]. The disease begins when the conidia of the pathogen come into contact with the hydrophobic surface of the rice leaf. These conidia then germinate and develop infection structures called appressoria. The appressoria accumulate high internal turgor pressure, allowing them to mechanically penetrate the rice cells by forming a hyphal penetration peg [2,3]. Once the pathogen has successfully colonized the rice tissue, multiple infection hyphae expand both within and between cells, leading to the formation of characteristic blast lesions within 3 to 5 days [4]. From these lesions, new conidia are produced and released, initiating new infection cycles. In addition to its impact on rice, M. oryzae can infect other important cereal crops such as wheat, barley, finger millet, and foxtail millet [5]. This fungus recently caused a devastating outbreak of wheat blast in Bangladesh, resulting in significant economic losses [6,7]. Due to its economic importance, genetic tractability, and the availability of its genome sequence, M. oryzae has emerged as a valuable model for studying fungal pathogenesis and its interaction with host plants [8,9]. Since TFs are considered pivotal regulators necessary for fitness and virulence in M. oryzae [10], further understanding of the cellular or biological functions mediated by these proteins should be beneficial for developing novel and practical strategies to control the blast disease and ensure global food security.
TFs are proteins that have DNA-binding domains. They can be classified into various families, including bZIP, bHLH, C2H2 zinc finger, homeobox, Zn2Cys6, and Myb, according to their specific DNA-binding domains [11]. There have been 495 predicted TFs identified in the M. oryzae genome and Myb family TFs belong to one of the major families [12]. Several of these identified TFs have been characterized and play different roles in the fungus, relating to its plant pathogenic lifestyle; conidiation [13,14,15,16]; appressorium formation [14,17,18]; host infection [14,19,20,21]; and, specifically in the early infection stage where a Zn2Cys6 TF is specifically activated, important for the progress of the infection, probably by regulating effectors necessary for entering the necrotrophic stage [22].
The Myb family is known for its highly conserved Myb DNA-binding domain. This domain typically consists of 1 to 3 imperfect amino acid repeats, each containing 50 to 53 amino acids, which form a helix-turn-helix structure [23,24,25,26,27]. The name “Myb” originated from v-Myb, the oncogenic motif of avian myeloblastosis virus (AMV), where it was initially identified [26]. The Myb family is present in all eukaryotes, including plants, animals, and fungi [28]. In plants, Myb family proteins are predominantly known as transcription factors or repressors [29,30,31,32,33,34,35]. However, proteins that are not classical transcription factors can contain Myb DNA binding domains [15,34,36,37,38,39,40,41,42,43]. In animal genomes, the Myb family is relatively small, consisting of only 4 to 5 proteins [24]. In contrast, the Myb family has expanded in plants, with a range of 100 to 200 members per genome [24]. The fungal Myb family size is smaller than in plants but more extensive than in animals, with 10 to 50 members per genome [11]. Animal Myb proteins have been reported to regulate cell division and a discrete subset of cellular differentiation events [24,44,45]. In plants, Myb proteins bind DNA and regulate many metabolic, cellular, and developmental processes as transcription factors [24,25,31,32,33,34,35]. Fungal Myb proteins have been implicated in stress response regulation and are thought to play a role in the pathogenicity of plant pathogens [46,47,48]. However, compared to Myb proteins in plants and animals, fungal Myb proteins have received less research attention, and their biological roles are largely unknown.
This research study aims to investigate the functional roles of the Myb family TFs in the rice blast fungus, M. oryzae. The Fungal Transcription Factor Database (FTFD, http://ftfd.snu.ac.kr/, accessed on 5 May 2022) predicted 19 Myb family TFs in M. oryzae. Before our study, one Myb TF was previously reported as MoMyb1 [49] and nine more recently identified as MoMyb2-10 [50]. In this study, we attempted to knock out three additional genes, with gene IDs MGG_05099, MGG_01130, and MGG_14558, which were respectively named MoMyb11-13. However, we were only successful in obtaining knockout mutants for MoMyb13. Therefore, this paper study has solely focused on investigating the function of MoMyb13. Our findings reveal that the MoMyb13 plays a key role for regulating the growth, conidiation, hydrophobin formation, and pathogenicity of M. oryzae.

2. Materials and Methods

Organisms and Media Used
Magnaporthe oryzae B. Couch anamorph of the teleomorph Pyricularia oryzae Cavara was used for this research. As background strain, we used Ku80 (generated from the WT strain 70-15) to minimize random integration events when transformed [51]. The susceptible Indica rice (cv. CO-39) and barley (cv. Golden Promise) used for the fungal pathogenicity tests were from the seed bank of our laboratory. The CM (complete medium), MM (minimal medium), and RBM (rice bran medium) used for growing the fungus were prepared as described [52]. The Escherichia coli strain DH-5α used for routine bacterial transformations and maintenance of various plasmid vectors was bought from Solarbio Life Sciences, Bejing, China. All primers used in this study are listed (Table S1).
Knockouts, Complementations, and Verifications
The MoMYB13 knockout vector was constructed in the plasmid pBS-HYG by inserting 1 kb up- and down-stream fragments of the MoMYB13 coding region as flanking regions of the HPH (hygromycin phosphotransferase) gene [53]. To perform gene deletion transformations, at least 2 μg of the vector DNA was introduced to Ku80 protoplasts, and transformants were selected for hygromycin resistance [54]. Southern blotting was conducted to confirm the correct knockout mutants using the digoxigenin (DIG) high prime DNA labelling and detection starter Kit I (11745832910, Roche, Mannheim, Germany). The complementation vector of MoMYB13 was constructed by cloning the entire length of MoMYB13 with the native promoter region (about 1.5 kb) to the pCB1532 plasmid. During the making of the complementation vector, GFP was linked to the C-terminal of MoMYB13 to study the sub-cellar localization of MoMyb13. The constructed vector DNA was introduced into the knockout mutant protoplast for gene complementation, and resulting transformants were screened using 50 μg/mL chlorimuron-ethyl to select successful complementary strains. The detailed fungal protoplast preparation and transformation methods have been described previously [55]. The sub-cellular localization of MoMyb13 was observed by confocal microscopy (Nikon A1, Tokyo, Japan). GFP and RFP excitation wavelengths were 488 nm and 561 nm, respectively.
Phenotypic Characterization of Mutant
Vegetative growth was tested by measuring the colony diameter after ten days of growth in 9 cm Petri dishes at 25 °C under 12 h-to-12 h light and dark periods. Conidia production was evaluated by flooding the 12-day-old colony with double distilled water, filtering out the mycelia with gauze, and counting the conidia using a hemacytometer (Z359629, Merck, Darmstadt, Germany). The conidiophore induction assay was performed by excising one thin agar block from the fungal colony and then incubating it in a sealed chamber for 24 h with constant light [56]. Hyphal appressoria formation was induced by placing a suspension of hyphal fragments on a hydrophobic surface in a humid environment at 25 °C for 24 h. Conidial appressoria was induced by placing a conidia suspension of 104 spores/mL in the same environment. The pathogenicity assay on excised barley and rice leaves was performed by cutting a small block from the agar culture of the fungus and placing it on excised leaves for five days in a moist chamber for disease development [57]. The wounded leaves were created by gently scraping the leaf surface at the inoculation site with a blade of a knife. Colony wettability was estimated and tested using a well-established wettability method used to demonstrate the absence of hydrophobin MPG1 [58].
qPCR assay
Total RNA was extracted using Eastep®Super Total RNA Extraction Kit (Promega (Beijing, China) Biotechnology, LS1040) to perform qPCR. A total of 5 μg of RNA (1 μg/μL) was reverse-transcribed to cDNA using the Evo M-MLV RT kit with gDNA to clean before the qPCR (Accurate Biotechnology (Changsha, China), AG11705) according to the manufacturer’s instructions. The resulting cDNA was diluted ten times and used as the template for the qPCR. The qPCR reactions were performed using an Applied Biosystems 7500 Real-Time PCR System. Each reaction contained 25 μL of SuperRealPreMix Plus SYBR Green (Tiangen Biotechnology, Beijing, China, FP205-02), 1.0 μL of cDNA, and 1.5 μL of each primer solution (0.5 μM/L). The thermal cycling conditions were 15 min at 95 °C followed by 40 cycles of 10 s at 95 °C and 20 s at 60 °C. The threshold cycle (Ct) values were obtained by analyzing amplification curves with a normalized reporter threshold of 0.1. The relative expression value was calculated using the 2−ΔΔCT method [59].

3. Results

3.1. Sequence Identification and Evolution Analysis of MoMyb13

According to the NCBI database, MoMyb13 is a large protein consisting of 2305 amino acids (aa) (Figure 1A). It has two Myb domains at positions 824-864 aa and 1119-1157 aa, and a transcription termination factor Rho domain at position 1849-2097 aa (Figure 1A). Additionally, it has two DNA polymerase III subunit γ/τ domains at 385-603 aa and 1192-1381 aa (Figure 1A). A similarity search conducted in the NCBI database (E value < 10−6) revealed that Momyb13 only has homologues in 17 genera of ascomycete fungi, but no homologues found in plants, animals, or fungi outside of the ascomycetes. We selected one homologue from each genus to construct a phylogenetic tree and found that Momyb13 is most closely related to the homologues from Diaporthaceae sp. (KAH8778409.1, full length) and Diaporthe eres (KAI7783256.1, full length) (Figure 1A).

3.2. Localization of MoMyb13 in the Nuclei of M. oryzae

Given that MoMyb13 is predicted to be a TF, we confirmed its nuclear localization through subcellular protein localization experiments. We fused the GFP protein to the C-terminus of MoMyb13 and co-transformed it with the nuclear marker protein Histon-RFP into M. oryzae. By observing the fluorescent signals of the transformants, we found a complete overlap of the red and green signals in the nuclei of hyphae, conidia, and appressoria of M. oryzae (Figure 2), indicating that MoMyb13 is also localized to the nucleus in M. oryzae, consistent with its role as a transcription factor.

3.3. MoMyb13 Is Required for the Growth of M. oryzae

To study the function of MoMyb13, we conducted gene knockout experiments and obtained two mutants, Δmomyb13-11 and Δmomyb13-16, which were confirmed by Southern blot (Figure S1). Since the two mutants have the same phenotypes, we only show the data of Δmomyb13-11. The complementary strain Δmomyb13-11/MoMYB13 was generated by reintroducing the MoMYB13 to Δmomyb13-11. Growth analysis of the mutant Δmomyb13-11 revealed a significant reduction in growth rate compared to the control strain ku80 and the complementary strain Δmomyb13-11/MoMYB13 on all three culture media, complete medium (CM), minimal medium (MM), and rice bran medium (RBM) (Figure 3A,B). Additionally, the mutant Δmomyb13-11 has a completely white colony color on CM indicating a lack of melanin pigments (Figure 3A). qPCR analysis further showed that two melanin pigment synthesis genes, MoBUF1 and MoALB1 [60], were significantly downregulated in the mutant Δmomyb13-11 (Figure S2), which explains the white colony of the MoMyb13 mutant. These results suggest that the MoMyb13 TF is required for regulating the vegetative growth and normal colony color development of M. oryzae.

3.4. MoMyb13 Is Essential for the Conidiogenesis of M. oryzae

Since conidia are essential for the fungus to spread disease, the conidiation of mutants was investigated [4]. We first observed the conidiophore formation, and found that the mutant only formed sparsely distributed conidiophores, whereas the control strain ku80 and complementary strain Δmomyb13-11/MoMYB13 exhibited dense conidiophores (Figure 4A). Statistical analysis of the number of conidia showed that the mutant did not produce any conidia on CM or RBM, while the control strain ku80 and complementary strain Δmomyb13-11/MoMYB13 produced a large number of conidia (Figure 4B). Determination of the expression of six conidiation-related genes revealed that three genes, MoCOS1, MoCON7, and MoCOM1, were significantly downregulated in the mutant (Figure S3). These results suggest that MoMyb13 is essential for the conidiation of M. oryzae.

3.5. MoMyb13 Is Essential for the Pathogenicity of M. oryzae

As M. oryzae is a pathogenic fungus, its pathogenicity is its most important property. Therefore, we analyzed the pathogenicity of the MoMyb13 mutant Δmomyb13-11. Since the mutant does not produce conidia, we used mycelial blocks for plant inoculation. This was done by placing the blocks onto detached plant leaves and incubating for 7 days. We found that the mutant completely lost pathogenicity to both barley and rice, while the control strain and complemented strain showed normal pathogenicity (Figure 5A,B). The mutant did not cause disease even on wounded leaves, (Figure 5A,B). Considering that the hyphae of M. oryzae can develop the infection structure, hyphal appressoria, we were particularly interested in whether the hyphae of the mutant could produce appressoria. By incubating crushed hyphae on a hydrophobic surface, we found that the mutant could produce hyphal appressoria normally (Figure 5C). However, since the mutant is unable to cause disease, these appressoria may be non-functional. These results indicate that MoMyb13 is essential for pathogenicity but is not involved in hyphal appressorium formation.

3.6. MoMyb13 Is Involved in Regulating Hydrophobins

The aerial mycelia of many ascomycetes are covered by small hydrophobic proteins, known as hydrophobins, making the colony hydrophobic [58,61]. Hydrophobins are also present in the aerial hyphae of basidiomycetes and they were first discovered and separated in the basidiomycete Schizophyllum commune [62]. We noticed that the MoMyb13 mutants had a more wettable colony [58] (Figure 6A), leading us to investigate if any hydrophobin genes were downregulated in the mutant. Our results showed that one of the four tested hydrophobin-related genes, MPG1 [63], was strongly downregulated (Figure 6B), prompting us to upregulate MPG1 in the MoMyb13 mutant (Figure 6C). However, the upregulation of MPG1 only restored the hydrophobicity of the mutant, but not the pathogenicity (Figure 6D). These results indicate that MoMyb13 regulates more genes necessary for rice pathogenicity than just hydrophobins.

3.7. MoMyb13 Is Involved in Stress Response

A stress assay was performed to assess the response of the MoMyb13 mutants to different stressors including sodium dodecyl sulfate (SDS), which affects membrane integrity; congo red (CR), which affects cell wall integrity; sodium chloride (NaCl), influencing ionic strength, water potential and potential sodium toxicity; sorbitol (SOR), affecting osmotic strength; and hydrogen peroxide (H2O2), inducing oxidative stress. The mutants displayed an increased inhibition rate to SDS, CR, and H2O2 (Figure 7A,B), indicating a high sensitivity to cell membrane and wall damage, and oxidative stress. Conversely, the mutants exhibited a decreased inhibition rate to NaCl and SOR (Figure 7A,B), suggesting a low sensitivity to salt and osmotic stress. Since melanin can act as a very good antioxidant [64], the diminished melanin resulted in a less dark coloring of the colonies [65] in the mutant (Figure 3A on CM medium especially) which may contribute to its increased sensitivity to oxidative stress.

4. Discussion

The MoMYB13 TF affected both growth and infection phenotypes although the effect seems to be limited to conidia formation and therefore conidia viability. This can be caused by the decreased content of dark melanin in the mutants’ mycelia and conidia (white mutant colonies) (Figure 3A) and also a lack of hydrophobicity of the conidia (Figure 6A). Melanin is an antioxidant which can protect the fungus from severe ROS (reactive oxygen species) stresses [64]. Conidial hydrophobicity affects the dispersion of dry conidia and their adhesion to the hydrophobic leaf surface of the host plants [58,63]. Thus, the MoMyb13 TF seems important for resisting stress during the necrotrophic phase and for the spread of viable conidia to new hosts. As mentioned in the introduction, other TFs have other roles during other stages of the infection. These roles were summarized in our previous paper where we described a TF with importance in the early infection in the transition between the short biotrophic phase to the longer necrotrophic phase [22]. The MoMyb13 protein is large (Figure 1A) compared to other Myb-proteins described for M. oryzae [50] and has no orthologs outside Ascomycota in the NCBI database. We investigated this further and found similarly large proteins with similar predicted aa sequences in 19 other published fungal genomes. Three of these hits were in different strains of Pyricularia oryzae (teleomorph of M. oryzae). Interestingly, common to all hits were that they were in fungal genomes of fungi known to produce melanized hydrophobic structures [66,67,68,69,70,71,72,73]. Our results (Figure 6) suggest a possible involvement of these similar proteins only found in Sordariomycetes in regulating hydrophobin and melanin production. Both MoMyb1 and MoMyb13 thus seem involved in these processes. However, artificial regulation of the main hydrophobin negatively affected by the mutation of MoMyb13 did not restore conidiation indicating that the mutation has affected more than just the formation of hydrophobins. Consequently, MoMyb13 does not regulate all melanin production in the fungus on its own, since appressorium formation still takes place in the mutant. Similarly, MoMyb1 appears to mainly regulate the MPG1 hydrophobin although no compensatory regulation was seen for Myb13 as for MoMyb1 [49].
The melanization of conidia and appressoria might be differently controlled. MoMyb1 is known to control HAD (hyphae-driven appressoria formation) melanization, and hydrophobin production [49]. This points to the conclusion that MoMyb1 and MoMyb13 might control melanization pigment formation differently in conidia and mycelia appressoria. Melanin production genes Alb1, Rsy1, and Buf1 have been extensively studied and these three genes are important for melanin production and appressoria formation in the strain 70-15 and Guy11 and the deletion of any of them stopped infection, even if some dark pigment was formed in the Buf1 mutant [60]. The main gene involved in producing pigmented colonies, Alb1, was downregulated in the MoMyb13 mutant which could also be the consequence of MoMyb13 regulating MoMyb1, which in turn regulates Alb1 (Figure S1). Melanin has important anti-oxidant properties [64] and seems essential for fungi to withstand high radiation and use radioactive mineral-rich particles as a source of mineral nutrients [74,75]. Ionizing radiation mainly generates hydroxyl radicals as a damaging agent [64]. Hydroxyl radicals are interestingly exploited by brown rotting fungi to degrade cellulose at a distance from the hyphae, so as to not to be affected themselves [76]. Fungal melanin formation is thus most likely an efficient strategy to withstand plant defenses producing all kinds of ROSs.
M. oryzae grows as a biotroph without killing the plant cells in the first stages of infection [3], which overlaps with the stages when MoMyb13 is specifically up-regulated. Interestingly, all the melanized fungi with orthologues to the MoMyb13 protein (Figure 1B) are known to grow only endophytically as biotrophs, or at least with an initial marked endophytic period [77] during infection with a biotrophy/necrotrophy transition [3]. Whether the orthologous genes in the other fungi have similar roles in the other fungi is unknown but could become a focus for future studies on the role of melanins and hydrophobins in plant infection of these other fungi with biotrophic initial stages.

5. Conclusions

Momyb13 is a TF with an important role in M. oryzae pathogenesis in rice. It regulates genes involved in necessary hydrophobin and melanin formation, important for conidial vitality, as well as the ROS stress resistance of mycelia. The encoding gene appears unique to dark fungi with biotrophic lifestyles or with the initial slightly extended biotrophic lifestyle of M. oryzae.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14020251/s1, Figure S1. Southern blot confirmation for the MoMyb13 knockout mutants. Figure S2. Bar chart showing the relative expression of three pigment related genes in MoMyb13 mutant Δmomyb13-11. The data shows the mean ± standard errors (SE, n = 3). The t-test was performed (p < 0.05). All the experiments were performed in replicates. Figure S3. Bar chart showing the relative expression of six conidition related genes in MoMyb13 mutant Δmomyb13-11. The data shows the mean + standard errors (SE, n = 3). The t-test was performed (p < 0.05). All the experiments were performed in replicates. Table S1. Primers used in this study.

Author Contributions

Y.L.: domain prediction and evolutionary analysis, gene deletion, phenotype tests, protein localization, qPCR, data collection and analysis, figure making, manuscript preparation, and writing. X.Z., MoMyb13 gene deletion, MoMyb13 mutant phenotype analysis, MoMyb13 protein localization, qPCR of hydrophobin gene. M.C., M.P., C.L., L.G., P.H. and S.Z., Myb-gene deletions. S.O. work connected with comparing the found Myb proteins with orthologues in other fungi, manuscript preparation, and writing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Fujian province (2022J01125), Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests (MIMCP-202301), Fujian Provincial Science and Technology Key Project (2022NZ030014) and the National Natural Science Foundation of China (NSFC31871914).

Data Availability Statement

Data supporting the findings of this study are available within the article or its Supplementary Materials.

Acknowledgments

The strain Ku80 used in this study was obtained initially from the lab of Nicholas J. Talbot, University of Exeter, UK.

Conflicts of Interest

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Domain identification and evolution analysis of MoMyb13. (A). The domain structures of Momyb13 were predicted by the NCBI database. The figure was drawn by the soft DOG 2.0. (B). Evolutionary relationships of MoMyb13 homologues were analyzed by MEGA7 using the Neighbor–Joining method. The MoMyb13 protein was in bold in the tree. The optimal tree with the sum of branch length = 4.48740141 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
Figure 1. Domain identification and evolution analysis of MoMyb13. (A). The domain structures of Momyb13 were predicted by the NCBI database. The figure was drawn by the soft DOG 2.0. (B). Evolutionary relationships of MoMyb13 homologues were analyzed by MEGA7 using the Neighbor–Joining method. The MoMyb13 protein was in bold in the tree. The optimal tree with the sum of branch length = 4.48740141 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
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Figure 2. MoMyb13-GFP colocalizes with Histon-RFP. The green signal of MoMyb13-GFP and red signal of Histon-RFP overlapped in the nuclei of hyphae, conidia, and appressoria of M. oryzae. The signals were tested by confocal microscopy (Nikon A1). The appressoria from the conidia was obtained by incubating the conidia on hydrophobic surfaces for 8 h at 25 °C. Bar, 5 μm.
Figure 2. MoMyb13-GFP colocalizes with Histon-RFP. The green signal of MoMyb13-GFP and red signal of Histon-RFP overlapped in the nuclei of hyphae, conidia, and appressoria of M. oryzae. The signals were tested by confocal microscopy (Nikon A1). The appressoria from the conidia was obtained by incubating the conidia on hydrophobic surfaces for 8 h at 25 °C. Bar, 5 μm.
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Figure 3. The MoMyb13 mutant Δmomyb13-11 was defective in growth and colony color. (A). Δmomyb13-11 showed reduced colony size on CM, MM, and RBM. The colony color of mutant on CM was completely white in comparison with Ku80 and Δmomyb13-11/MoMYB13, showing a lack of the dark melanin pigment. (B). Bar chart analysis of the Δmomyb13-11 growth rate. The data indicate the colony diameter of each strain growing for 7 days, showing mean + standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates.
Figure 3. The MoMyb13 mutant Δmomyb13-11 was defective in growth and colony color. (A). Δmomyb13-11 showed reduced colony size on CM, MM, and RBM. The colony color of mutant on CM was completely white in comparison with Ku80 and Δmomyb13-11/MoMYB13, showing a lack of the dark melanin pigment. (B). Bar chart analysis of the Δmomyb13-11 growth rate. The data indicate the colony diameter of each strain growing for 7 days, showing mean + standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates.
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Figure 4. The MoMyb13 mutant Δmomyb13-11 produced no conidia. (A). Δmomyb13-11 formed sparsely distributed conidiophores. Bar, 50 μm. (B). Bar chart analysis of conidial production. The data indicate the conidial number of each strain growing for 12 days in a 9 cm plate, showing the mean + standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates. * indicates the sign of multiplication.
Figure 4. The MoMyb13 mutant Δmomyb13-11 produced no conidia. (A). Δmomyb13-11 formed sparsely distributed conidiophores. Bar, 50 μm. (B). Bar chart analysis of conidial production. The data indicate the conidial number of each strain growing for 12 days in a 9 cm plate, showing the mean + standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates. * indicates the sign of multiplication.
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Figure 5. The MoMyb13 mutant Δmomyb13-11 lost pathogenicity but produced hyphal appressoria. (A) Δmomyb13-11 is unable to cause lesions on both normal and wounded leaves of barley. (B) Δmomyb13-11 is unable to cause lesions on both normal and wounded leaves of rice. (C) The hyphae of Δmomyb13-11 were able to produce hyphal appressoria as normally as in the control strain ku80 and the complementary strain Δmomyb13-11/MoMYB13. Bar, 20 μm.
Figure 5. The MoMyb13 mutant Δmomyb13-11 lost pathogenicity but produced hyphal appressoria. (A) Δmomyb13-11 is unable to cause lesions on both normal and wounded leaves of barley. (B) Δmomyb13-11 is unable to cause lesions on both normal and wounded leaves of rice. (C) The hyphae of Δmomyb13-11 were able to produce hyphal appressoria as normally as in the control strain ku80 and the complementary strain Δmomyb13-11/MoMYB13. Bar, 20 μm.
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Figure 6. The MoMyb13 mutant Δmomyb13-11 has a wettable phenotype with decreased hydrophobin production. (A) Colonies of Δmomyb13 are easily wettable compared to the surface of the control strain (Ku80), the complementary strain, or the Δmomyb13 strain containing the overexpressed hydrophobin MPG1 (Δmomyb13-11/TrpC-MPG1 is not wettable). (B) Percent change in expression of 4 hydrophobin genes in Δmomyb13 compared to Ku80 shows that MPG1 is severely downregulated while the other hydrophobins are less affected. (C) Percent change in MPG1 expression in Ku80, Δmomyb13, and Δmomyb13/TrpC-MPG1 strains compared to Ku80. (D) Leaf lesions formed by Ku80, Δmomyb13, and Δmomyb13/TrpC-MPG1 strains using the agar block inoculation. Error bars (B,C) show 95% confidence intervals. Bars with confidence intervals not overlapping with other bars’ confidence intervals or levels (as zero) are significantly different (p < 0.05 for the null hypothesis that they are the same). All the experiments were performed in replicates.
Figure 6. The MoMyb13 mutant Δmomyb13-11 has a wettable phenotype with decreased hydrophobin production. (A) Colonies of Δmomyb13 are easily wettable compared to the surface of the control strain (Ku80), the complementary strain, or the Δmomyb13 strain containing the overexpressed hydrophobin MPG1 (Δmomyb13-11/TrpC-MPG1 is not wettable). (B) Percent change in expression of 4 hydrophobin genes in Δmomyb13 compared to Ku80 shows that MPG1 is severely downregulated while the other hydrophobins are less affected. (C) Percent change in MPG1 expression in Ku80, Δmomyb13, and Δmomyb13/TrpC-MPG1 strains compared to Ku80. (D) Leaf lesions formed by Ku80, Δmomyb13, and Δmomyb13/TrpC-MPG1 strains using the agar block inoculation. Error bars (B,C) show 95% confidence intervals. Bars with confidence intervals not overlapping with other bars’ confidence intervals or levels (as zero) are significantly different (p < 0.05 for the null hypothesis that they are the same). All the experiments were performed in replicates.
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Figure 7. The MoMyb13 mutant Δmomyb13-11 showed responses to different stress. (A) Colony size and morphology of the mutant Δmomyb13-11, control strain ku80 and complementary strain Δmomyb13-11/MoMYB13 after 10 days growing on CM or CM with additions of SDS, CR, NaCl SOR, or H2O2 as stresses (see methods). (B) Bar chart analysis of the sensitivity of Δmomyb13-11 to different stressors. The data indicate the growth inhibition rate of each strain growing on CM with stressors, showing the mean ± standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates.
Figure 7. The MoMyb13 mutant Δmomyb13-11 showed responses to different stress. (A) Colony size and morphology of the mutant Δmomyb13-11, control strain ku80 and complementary strain Δmomyb13-11/MoMYB13 after 10 days growing on CM or CM with additions of SDS, CR, NaCl SOR, or H2O2 as stresses (see methods). (B) Bar chart analysis of the sensitivity of Δmomyb13-11 to different stressors. The data indicate the growth inhibition rate of each strain growing on CM with stressors, showing the mean ± standard errors (SE, n = 3). The t-test was performed (p < 0.05) and bars with the same color but with different letters are significantly different. All the experiments were performed in replicates.
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Li, Y.; Zheng, X.; Pei, M.; Chen, M.; Zhang, S.; Liang, C.; Gao, L.; Huang, P.; Olsson, S. Functional Analysis of MoMyb13, a Myb Transcription Factor Involved in Regulating Growth, Conidiation, Hydrophobicity, and Pathogenicity of Magnaporthe oryzae. Agronomy 2024, 14, 251. https://doi.org/10.3390/agronomy14020251

AMA Style

Li Y, Zheng X, Pei M, Chen M, Zhang S, Liang C, Gao L, Huang P, Olsson S. Functional Analysis of MoMyb13, a Myb Transcription Factor Involved in Regulating Growth, Conidiation, Hydrophobicity, and Pathogenicity of Magnaporthe oryzae. Agronomy. 2024; 14(2):251. https://doi.org/10.3390/agronomy14020251

Chicago/Turabian Style

Li, Ya, Xiuxia Zheng, Mengtian Pei, Mengting Chen, Shengnan Zhang, Chenyu Liang, Luyao Gao, Pin Huang, and Stefan Olsson. 2024. "Functional Analysis of MoMyb13, a Myb Transcription Factor Involved in Regulating Growth, Conidiation, Hydrophobicity, and Pathogenicity of Magnaporthe oryzae" Agronomy 14, no. 2: 251. https://doi.org/10.3390/agronomy14020251

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