Identification and Functional Characterization of the Heat Shock Protein (HSP) 40 Encoding Gene, MoMHF6, in Magnaporthe oryzae

doi:10.16819/j.1001-7216.2023.230301

Abstract

Abstract:

【Objective】 To explore the roles of chaperone HSP40 (heat shock protein 40) in morphological differentiation and pathogenesis in Magnaporthe oryzae. 【Method】 The HSP40 encoding gene MoMHF6 of M. oryzae was knocked out by DNA homologous recombination, and a deletion mutant ΔMomhf6 was generated. Biological function of the gene was systematically characterized via phenotypic analysis, gene complementation and RNA-seq analysis. 【Result】 Phenotypic analysis of the mutant revealed severe impairments in aerial hypha growth, asexual sporulation, conidial germination, perithecium production and appressorium formation. However, compared to the wild-type strain, the deletion of MoMHF6 did not affect mycelium radial growth on CM medium and the development of asci and ascospores in perithecia. Incipient cytorrhysis assays revealed that the appressorium collapse rate of the ΔMomhf6 mutant under treatment with 1, 2 or 3 mol/L glycerol was significantly increased compared to the wild-type strain, indicating that MoMHF6 is required for appressorium turgor generation. Also, we found that the ΔMomhf6 mutant was unable to penetrate into onion epidermis and completely nonpathogenic to susceptible hosts. Even in wounded barley leaves, infectious growth of the mutant was severely impaired. Moreover, the deletion of MoMHF6 resulted in significantly increased sensitivity to oxidative stress and delayed glycogen transportation and degradation during appressorium development, indicating that MoMHF6 is involved in oxidative stress response and glycogen metabolism of appressorium. All phenotypic defects of the ΔMomhf6 mutant could be restored by reintroducing the full-length MoMHF6 gene into the mutant. In addition, RNA-seq analysis of the ΔMomhf6 mutant revealed that several known pathogenicity-related genes in differentially expressed genes (DEGs) might be regulated by MoMHF6, such as MoATG4, MoPL1, MoVPR, and GAS1. 【Conclusion】 The HSP40 gene MoMHF6 plays an important role in asexual sporulation, appressorium formation, host penetration, oxidative stress response and pathogenicity in M. oryzae. These results are of great significance for further understanding the gene networks and molecular mechanisms in regulating morphological differentiation and pathogenesis governed by MoMHF6 in M. oryzae.

Key words: Magnaporthe oryzae, MoMHF6, heat shock protein 40, functional analysis, RNA-seq

摘要：

【目的】 探明稻瘟病菌热激蛋白（HSP）40在形态分化和致病过程中的作用。【方法】 利用DNA同源重组方法敲除了稻瘟病菌HSP40编码基因MoMHF6，获得ΔMomhf6突变体，并通过表型分析、基因回补、RNA-seq分析等对MoMHF6的生物学功能进行较系统的研究。【结果】 敲除MoMHF6基因导致稻瘟病菌气生菌丝生长、无性产孢、孢子萌发、子囊壳产生和附着胞形成均显著下降，但与野生菌株相比，ΔMomhf6突变体在CM培养基上的径向生长没有显著差异，在子囊壳中仍可形成子囊和子囊孢子。在1、2和3 mol/L甘油溶液处理条件下，ΔMomhf6突变体附着胞塌陷率显著升高，说明MoMHF6与附着胞膨压形成有关。ΔMomhf6突变体对洋葱内表皮的穿透能力和对感病寄主的致病性完全丧失，甚至在划伤大麦叶片表皮组织中的扩展也受明显限制。而且，ΔMomhf6突变体对氧化胁迫的敏感性增强，附着胞发育过程中糖原的转运和降解缓慢，说明MoMHF6参与氧化胁迫反应和附着胞的糖原代谢。将完整的MoMHF6基因重新导入ΔMomhf6突变体可回补突变体的所有表型缺陷。另外，RNA-seq分析显示，部分已知的稻瘟病菌致病相关基因表达可能受MoMHF6调控，如MoATG4、MoPL1、MoVPR和GAS1等。【结论】 综上所述，稻瘟病菌HSP40编码基因MoMHF6在产孢、附着胞形成、穿透寄主、氧化胁迫应答、致病等过程中起重要作用，研究结果对进一步阐明MoMHF6调控稻瘟病菌形态分化和致病过程的基因网络和分子机制具有重要意义。

关键词: 稻瘟病菌, MoMHF6, 热激蛋白40, 功能分析, RNA-seq

TONG Qi, WANG Chunyan, QUE Yawei, XIAO Yu, WANG Zhengyi. Identification and Functional Characterization of the Heat Shock Protein (HSP) 40 Encoding Gene, MoMHF6, in Magnaporthe oryzae[J]. Chinese Journal OF Rice Science, 2023, 37(6): 563-576.

童琪, 王春燕, 阙亚伟, 肖宇, 王政逸. 稻瘟病菌热激蛋白(HSP)40编码基因MoMHF6的鉴定及功能研究[J]. 中国水稻科学, 2023, 37(6): 563-576.

Figures/Tables 10

Table 1. Primers used in this study.

引物 Primer	序列 Sequence (5′-3′)
MHF6-up-F	CCCCCGGGCTGCAGGAATTCGAGATAACAAAAGGTAT
MHF6-up-R	GCTCCTTCAATATCATCTTCTCTCGCTCAGTTCGAAATGGGAT
MHF6-down-F	TAGAGTAGATGCCGACCGAACAAGAATCAATGCCCAGTCTCGTGC
MHF6-down-R	TACCGGGCCCCCCCTCGAGCGAATACACTTTGGAGAC
HPT-F	GACAGACGTCGCGGTGAGTT
HPT-R	GTCCGAGGGCAAAGAAATAG
MHF6-YW-F	TCGAGGAGATTGAGTGCGTC
MHF6-YW-R	CTTCTTTTGCTTGGCTTTGC
MHF6-DX-F	GACTTCAATCAACCCTAACC
MHF6-DX-R	AAGCCGGACGGAAAGACTTT
HB-MHF6-F	TCCCCCGGGCTGCAGGAATTCTTCTCCAGAAAATCCCTGGA
HB-MHF6-R	GATAAGCTTGATATCGAATTCATTCGACCGGCGATCTTCCG
Actin-RT-F	ATTTACGAGGGTTTCTCCTTGC
Actin-RT-R	TCTCCTGCTCAAAGTCAAGAG
HOX2-qRT-F	CGATAATTGCTCCCACACCT
HOX2-qRT-R	GAAGGAGTCGGTGGTGACAT
COS1-qRT-F	ATGGATTCCCAGCCTCGTA
COS1-qRT-R	CGTTGACCAGCAAAGACAA
HTF1-qRT-F	GGCGACGATACGAAGAAA
HTF1-qRT-R	TGAACCACCTTGGCTTTG
CON7-qRT-F	GGCGACGATACGAAGAAA
CON7-qRT-R	TGAACCACCTTGGCTTTG
COM1-qRT-F	GAAAGAACCTATCAGGGCG
COM1-qRT-R	GTTTGCGATTGGCATTAGC
STU1-qRT-F	CTACGTTAAGTCCGAGATGG
STU1-qRT-R	CGTGATCAGCCTCATCTTCC

Fig. 1. Phylogenetic analysis of MoMhf6 and its orthologues from several fungal species. The phylogenetic tree was constructed by MEGA version 7.0. Numbers at nodes of the branch represent bootstrapping value in 1000 replications. The length of the branches represents genetic distance. The distance scale=0.05.

Fig. 2. Schematic diagram of gene knockout of MoMHF6 and confirmation of ΔMomhf6 mutants and complemented transformants by PCR. A, Knockout vector pKO-MoMHF6 and target gene deletion of MoMHF6. The red arrow represents the target gene MoMHF6. The blue arrow represents the hygromycin resistant gene. The location of primers is indicated; B, Confirmation of the deletion mutant ΔMomhf6 and the complemented strain ΔMomhf6-C. Left: A 524 bp MoMHF6 gene fragment was amplified by primers MHF6-YW-F/R. Right: the targeted gene deletion or complementation events of different strains were further verified by primers MHF6-DX-F/R. The bands of about 1.5 kb and 3.14 kb were corresponding with HPH and MoMHF6 genes respectively. M, Marker; Lane 1, Guy11; Lane 2, ΔMomhf6; Lane 3, MoMHF6-ect (ectopic); Lane 4, ΔMomhf6-C (complemented).

Fig. 3. MoMHF6 is required for aerial hypha growth and perithecia production. A, The wild type Guy11, the ΔMomhf6 mutant, the ectopic insertion strain MoMHF6-ect and the complemented strain ΔMomhf6-C were cultured on CM plates at 25 ℃ for 10 days and photographed at 10 days after inoculation; B, The bar graph shows radical growth of different strains on CM plates. Error bars represent standard error; C, Perithecia production was severely reduced for the ΔMomhf6×TH3 cross. Asci and ascospores in perithecia of both crosses were observed. Bar=20 μm.

Fig. 4. MoMHF6 plays important roles in pathogenesis by M. oryzae. A, The ΔMomhf6 mutant is nonpathogenic to susceptible barley leaves. Conidial suspension (5×104 conidia/mL) of different tested strains were drop-inoculated onto barley leaf segments and photographed at 5 dpi (days post inoculation); B, The ΔMomhf6 mutant is nonpathogenic to susceptible rice leaves. Rice seedlings were sprayed with conidial suspension (5×104 conidia/mL) of different strains and photographed at 5 dpi; C, Invasive growth of the ΔMomhf6 mutant was severely impaired on wounded barley leaf segments. Conidial suspension (5×104 conidia/mL) of different strains was dropped on wounded barley leaf segments. CK represents H2O. Photographs were taken at 5 dpi; D, The ΔMomhf6 mutant was unable to penetrate onion cuticle and cell wall. Conidial suspension (2×104 conidia/mL) of different strains was dropped on onion epidermis. After 24 h and 48 h of incubation in darkness, the onion epidermis was examined and photographed under a light microscope. Bars=20 μm.

Fig. 5. MoMHF6 is important for asexual sporulation. A, Microscopic observation of aerial hypha growth and conidiophore development. Bars=100 μm; B, Statistical analysis of conidiation on CM at 25 ℃ for 12 days; C, qRT-PCR analysis of transcriptional expression of several sporulation related genes. Error bars represent standard error. Asterisks indicate significant difference (*P<0.05; **P<0.01).

Fig. 6. Deletion of MoMHF6 results in significant reduction of conidium germination, appressorium formation and appressorium turgor generation A, Statistical analysis of conidium germination. Conidia were allowed to germinate on hydrophobic surface for 6 hours; B, Statistical analysis of appressorium formation. Conidia were placed on the hydrophobic surface to induce appressorium formation and incubated for 24 hours; C, Statistical analysis of collapsed appressoria. Appressoria induced for 24 hours were incubated in 1 mol/L, 2 mol/L or 3 mol/L glycerol solution for 10 minutes. Error bars represent standard error. Asterisks indicate significant difference (*P<0.05; **P<0.01).

Fig. 7. Deletion of MoMHF6 delays glycogen transportation and degradation during appressoria development in M. oryzae. A, Conidial suspension (5×104 conidia/mL) of the wild type Guy11, the mutant ΔMomhf6 and the complemented strain ΔMomhf6-C was incubated on hydrophobic surface for 4, 8, 12, 24 hours respectively and then stained with KI/I2 solution. Photographs were taken at different time intervals. Bars=20 μm. B, Statistical analysis of glycogen mobilization in conidia of different tested strains; C, Statistical analysis of glycogen degradation in appressoria of different tested strains. Error bars represent standard error. Asterisks indicate significant difference (P<0.05).

Fig. 8. Deletion of MoMHF6 increases sensitivity of M. oryzae to oxidative stress. A, The different tested strains were inoculated on CM supplemented with 2.5, 5 and 10 mmol/L H2O2, respectively. Growth of various tested strains after 10 days is presented; B, Colony diameters were measured and growth inhibition rates were calculated. Error bars represent standard deviation. Asterisks indicate significant difference (*P<0.05; **P<0.01).

Fig. 9. Transcriptome analysis of the ΔMomhf6 mutant for differentially expressed genes. A, Statistical analysis of numbers of differentially expressed genes in the ΔMomhf6 mutant in comparison to the wild type strain Guy11; B, Scatter plot of GO term enrichment of DEGs in the ΔMomhf6 mutant. Rich ratio represents the ratio of numbers of DEGs annotated in the GO term to the numbers of all genes annotated in the same GO term. The q-value is corrected p value, with lower value means greater intensiveness.

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