探討番茄萎凋病菌鈣調磷酸酶於菌絲生長及致病性所扮演之角色

Abstract

鐮孢菌(Fusarium)為絲狀真菌，包含許多重要的植物病原菌、真菌毒素(mycotoxin)生產者以及潛在的人類病原。尖鐮孢菌(F. oxysporum)可感染超過100種不同的作物造成嚴重危害且會在免疫缺乏個體(Immunocompromised individuals)造成系統性的鐮孢菌病症。鈣調磷酸酶(calcineurin)對許多病原真菌的生長及致病力扮演重要角色，但在不同的真菌，如人類病原真菌Candida albicans及Cryptococcus neoformans，或是植物病原真菌Magnaporthe oryzae和Ustilago maydis則有調控上的差異，例如造成對不同藥物和環境因子(溫度和酸鹼值)的敏感性差異。醫藥上，免疫抑制藥物FK506 和cyclosporine A為calcineurin抑制劑，與其他藥物如azoles或echinocandins綜合使用能有效對抗具耐藥性的真菌病原並使calcineurin成為很好的抗真菌藥物標靶。Calcineurin是由catalytic (Cna1)和regulatory (Cnb1) subunits組成，且為heterodimeric calmodulin-dependent的蛋白磷酸酶，其可調控病原真菌的Ca2+ 訊息傳遞、型態發育、環境壓力反應及致病力。然而calcineurin在尖鐮孢菌所扮演的角色仍未有研究報導。本研究以番茄萎凋病菌(F. oxysporum f. sp. lycopersici)為主軸，根據序列比對多種真菌的calcineurin，我們發現番茄萎凋病菌Cna1次單元中的真菌所特有之序列serine-proline rich region (SPRR)與其他真菌呈現差異。目前研究結果顯示突變株Δcna1和Δcnb1的蛋白磷酸酶活性測試分別為110.09±56.01和90.17±7.34，顯著低於野生株645.08±49.34 nmoles．min-1．mg-1 (p<0.001)。另外，Δcna1與 Δcnb1突變株菌絲生長速率分別為3.87±1.03 μm/hr和3.49±0.68 μm/hr，皆比野生株28.95±8.79 μm/hr慢約7倍；菌絲之隔膜間距分別為67.87±15.51 μm和72.69±14.01 μm為野生株隔膜間距167.95±47.76的一半，暗示隔膜之形成可能影響突變株菌絲生長速率。進一步測量Δcna1、Δcnb1突變株於28 oC之孢子萌發時間分別為10.8±0.5 hr和10.8±0.5 hr，皆比野生株6.3 ± 0.7 hr慢約4小時。另外兩突變株於100 mL PDB液態培養7天之產孢數量除以菌絲乾重為3.42±1.65×106 CFU/g及5.57±1.88×106 CFU/g顯著低於野生株產孢量9.8±4.09×107 CFU/g (p<0.05)；而Δcna1與Δcnb1突變株於100 mL芹菜培養基(20% celery + 0.05M Na2SO4)液態培養14天之厚膜孢子數量除以菌絲乾重約為4.17±1.39×105 CFU/g和2.62±1.25×105 CFU/g顯著低於野生株厚膜孢子形成數量1.62±0.78×107 CFU/g (p<0.05)。值得注意的是我們發現兩突變株於液態培養時會有絮凝現象(flocculation)的發生，而兩突變株之互補株Δcna1+CNA1和Δcnb1+CNB1皆回復近似野生株之表現型。另外也發現Δcna1與Δcnb1突變株對番茄農友301品系喪失致病力，而簡易萃取檢測Δcna1發現真菌毒素 Moniliformin和Fumonisin B1生產量降低。未來研究將可以朝向探討入侵菌絲的穿透能力、多樣的環境壓力測試及利用 RNA-sequencing與phosphoproteomics分別尋找相關的下游基因及受質(substrates)，並期望針對尖鐮孢菌calcineurin調控菌絲生長及致病機制提供更進一步的闡明。

Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. The soil-borne ascomycete Fusarium oxysporum attacks over 100 different crops and can cause systemic fusariosis in immunocompromised individuals. Previously reports suggested that calcineurin pathway governs the growth and virulence of multiple pathogenic fungi that infect humans (Candida albicans and Cryptococcus neoformans) or plants (Magnaporthe oryzae and Ustilago maydis). These studies also revealed that calcineurin regulates fungal Ca2+ signaling, morphogenesis, stress responses and drug tolerance. However, the roles of calcineurin in hyphal growth and virulence are still unknown in F. oxysporum. In medicine, the immunosuppressive calcineurin inhibitors (FK506 and cyclosporine A) are active against fungi alone or in combination with antifungal drugs, including azoles or echinocandins and renders these drugs fungicidal, even towards drug-resistant strains, making calcineurin a promising antifungal drug target. Calcineurin is a heterodimeric calmodulin (CaM)-dependent protein phosphatase comprised of catalytic (Cna1) and regulatory (Cnb1) subunits. In phylogenetic analysis, we found that Fusarium oxysporum calcineurins are diverged with very tiny intraspecific variation but districted to yeasts and other filamentous fungi. In this work, we obtain calcineurin deletion mutants (Δcna1 and Δcnb1) and complementary strain (Δcna1+CNA1 and Δcnb1+CNB1) in F. oxysporum f. sp. lycopersici. Our results demonstrate that phosphatase activity of Δcna1 (110.09±56.01 nmoles．min-1．mg-1) and Δcnb1 (90.17±7.34 nmoles．min-1．mg-1) are significant lower than the wild-type 4287 (645.08±49.34 nmoles．min-1．mg-1) (p<0.001). Compare to the wild-type (28.95±8.79 μm/hr), Δcna1 (3.87±1.03 μm/hr) and Δcnb1 (3.49±0.68 μm/hr) exhibit about 7 times slower hyphal growth rates. Meanwhile, interval of septum of Δcna1 (67.87±15.51 μm) and Δcnb1 (72.69±14.01 μm) are about half of the length of wild-type (167.95±47.76 μm). These results indicate that septum formation may give a great impact on hyphal growth rate in F. oxysporum f. sp. lycopersici. Furthermore, germination time of Δcna1 (10.8±0.5 hr) and Δcnb1 (10.8±0.5 hr) is 4 hr longer than the wild-type (6.3 ± 0.7 hr). Statistical analysis of conidiation in 100 mL PDB culture at 28 oC for 7 days, numbers of conidia in Δcna1 (3.42±1.65×106 CFU/g) and Δcnb1 (5.57±1.88×106 CFU/g) are less than the wild-type (9.8±4.09×107 CFU/g) (p<0.05). Besides, numbers of chlamydospore formation are also quantified, Δcna1 (4.17±1.39×105 CFU/g) and Δcnb1 (2.62±1.25×105 CFU/g) exhibit impaired development of chlamydospore formation in contrast to the wild-type (1.62±0.78×107 CFU/g) (p<0.05). It is worthy to notice that Δcna1 and Δcnb1 not only show flocculation in liquid culture without changing their hydrophobicity of colonies but also lose their pathogenicity on tomato host. Meanwhile, the complementary strains restore the wild-type like phenotype and pathogenicity. In mycelium crude extraction by acetonitrile/methanol/water (16:3:1; v/v/v), Δcna1 seems to have less pigment and also imply that the production of Moniliformin and Fumonisin B1 is decreased. From these phenotypic results, we can investigate further related to the roles of calcineurin pathway and utilizing RNA-sequencing and phosphoproteomics approaches to downstream gene and substrates, respectively.