自噬相关基因FpAtg3参与假禾谷镰孢的生长和致病

doi:10.3864/j.issn.0578-1752.2024.06.005

摘要/Abstract

摘要：

【背景】假禾谷镰孢（Fusarium pseudograminearum）引起的小麦茎基腐病是我国小麦生产上的新病害。自噬是真核生物中普遍发生的细胞过程，调控多种植物病原真菌的生长发育和侵染，但其在假禾谷镰孢中的作用还不明确。【目的】明确假禾谷镰孢自噬相关基因FpAtg3在该病菌生长和致病中的作用，解析假禾谷镰孢致病机理，为小麦茎基腐病科学防控提供理论依据。【方法】从NCBI数据库中下载主要真菌的Atg3氨基酸序列，利用MEGA5.05构建系统进化树。利用Split-PCR策略构建FpAtg3基因敲除盒，经PEG介导的原生质体转化导入假禾谷镰孢野生型菌株中。利用潮霉素抗性筛选阳性转化子，并经PCR检测获得FpAtg3基因缺失突变体（ΔFpAtg3）。构建pKNTG-FpAtg3重组载体，导入ΔFpAtg3菌株，利用FpAtg3自身启动子启动FpAtg3的转录，以获得FpAtg3缺失突变体的回补菌株。利用假禾谷镰孢营养生长的培养基PDA、CM和MM测定菌丝生长速率和菌落形态；PDA培养基上测定菌丝形态和菌丝融合率；CMC培养液中测定分生孢子的产量及形态；皮氏培养基上测定孢子融合芽管调控的融合率。将假禾谷镰孢的菌丝块接种小麦胚芽鞘和大麦叶片测定病原菌的致病力，采用盆栽试验测定假禾谷镰孢引起的小麦茎基腐病。在PDA培养基中分别加入刚果红、SDS和过氧化氢测定假禾谷镰孢对细胞壁、细胞膜和氧化胁迫的耐受性。【结果】细胞自噬相关蛋白Atg3在真菌中非常保守，且与生物进化的方向一致，假禾谷镰孢FpAtg3与禾谷镰孢和尖镰孢的Atg3同源性最高。获得了FpAtg3基因缺失突变体和回补菌株，表型测定结果显示，与假禾谷镰孢野生型和回补菌株相比，ΔFpAtg3在营养培养基上的菌丝生长明显减慢，气生菌丝减少，菌丝呈波纹状卷曲，分生孢子产量减少，孢子变短，分生孢子分隔减少；野生型和回补菌株菌丝融合发生非常普遍，在相同条件下ΔFpAtg3的菌丝融合率和孢子融合芽管调控的融合率均明显降低；ΔFpAtg3侵染大麦叶片和小麦胚芽鞘造成的病斑较野生型和回补菌株侵染的病斑明显减小，盆栽试验进一步验证ΔFpAtg3的致病力降低；与野生型和回补菌株相比，ΔFpAtg3在刚果红、SDS和过氧化氢胁迫中的耐受性降低。【结论】自噬相关基因FpAtg3参与假禾谷镰孢的菌丝生长、分生孢子产生、菌丝融合、致病力以及对非生物胁迫的耐受性。

关键词: 小麦茎基腐病, 假禾谷镰孢, Atg3, 菌丝融合, 致病力

Abstract:

【Background】 Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is one of the newly-occurred destructive diseases in the main wheat-growing region of China. Autophagy is an evolutionarily conserved cellular process that regulates the growth, development and infection of different phytopathogenic fungi. However, the role of autophagy in F. pseudograminearum is still unknown.【Objective】 To clarify the role of the autophagy gene FpAtg3 in the growth and pathogenicity of F. pseudograminearum, understand the mechanisms of the fungal infection, and to provide a theoretical basis for FCR prevention.【Method】 The known Atg3 proteins of different fungi were downloaded from NCBI, and the MEGA 5.05 was used to construct the phylogenetic tree of Atg3 proteins. The Split-PCR approach was used to generate FpAtg3 gene-replacement constructs, and then the FpAtg3 deletion mutants (ΔFpAtg3) were constructed by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and obtained by hygromycin resistance screening and PCR detection. The sequence of FpAtg3 and its native promoter was amplified and fused in pKNTG vector. The plasmid of pKNTG-FpAtg3 was then introduced into ΔFpAtg3 protoplasts for the complementation assay. The hyphal growth and colony morphology of the wild type, ΔFpAtg3 and the complementary (FpAtg3-C) strains were assayed on PDA, CM and MM plates. The hyphal blocks were cultured on PDA plates for hyphal morphology and hyphal fusion test; hyphal blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured on Petri dishes to explore conidial anastomosis tube (CAT)-mediated fusion. The hyphal blocks were inoculated on wheat coleoptiles and barley leaves to explore pathogenicity, and the pot-culture experiment was performed for FCR assay. Congo red, sodium dodecyl sulphate (SDS) and H₂O₂ reagents were added to PDA plates to determine the F. pseudograminearum responses to cell wall, cell membrane and oxidative stresses, respectively.【Result】 Atg3 homologous proteins from different fungi were highly conserved and consistent with the direction of species evolution. The FpAtg3 had a close relationship with Atg3 of Fusarium graminearum and Fusarium oxysporum. The ΔFpAtg3 and FpAtg3-C strains were obtained. The phenotypic measurements showed that compared to wild type and FpAtg3-C strains, ΔFpAtg3 exhibited significantly reduced colony growth rates and aerial hypha, curved hypha, reduced conidiation, shorter in length and fewer in septa; ΔFpAtg3 also showed significantly reduced hyphal fusion rate and CAT-mediated fusion rate. The pathogenicity of ΔFpAtg3 on wheat coleoptiles and barley leaves was significantly reduced comparing to that of the wild type and FpAtg3-C strains. The reduced pathogenicity of ΔFpAtg3 was further examined on FCR. Furthermore, ΔFpAtg3 displayed more sensitive to cell wall, cell membrane and oxidative stress than that of the wild type and FpAtg3-C strains.【Conclusion】 Autophagy-related gene FpAtg3 plays important roles in growth, conidiation, hyphal fusion, pathogenicity and response to abiotic stresses of F. pseudograminearum.

Key words: Fusarium crown rot (FCR), Fusarium pseudograminearum, Atg3, hyphal fusion, pathogenicity

董在芳, 丁腾腾, 单艺轩, 李洪连, 陈琳琳, 邢小萍. 自噬相关基因FpAtg3参与假禾谷镰孢的生长和致病[J]. 中国农业科学, 2024, 57(6): 1080-1090.

DONG ZaiFang, DING TengTeng, SHAN YiXuan, LI HongLian, CHEN LinLin, XING XiaoPing. Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum[J]. Scientia Agricultura Sinica, 2024, 57(6): 1080-1090.

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引物Primer	序列Sequence (5′-3′)
FpAtg3-F1	TGATAAGAAAGAGCAAGCT
FpAtg3-R1	TTGACCTCCACTAGCTCCAGCCGATGGCTTCGGGATTGATAA
FpAtg3-F2	ATAGAGTAGATGCCGACCGCGGGTTCACTGCATGGCTTATC
FpAtg3-R2	GCCTGGTTCCTGATCTAGAT
FpAtg3-PF	CTTCCAAAGACCTGACTAC
FpAtg3-PR	GGCCCCTTCAACGTTCTAG
FpAtg3nj-F	GCCAAATCACACCCGAAGAG
FpAtg3nj-R	GAATCGGCGCCAGAATCTC
HYG/F	GTCGACAGAAGATGATATTG
HYG/R	GAACCCGCGGTCGGCATCTACTCTAT
YG/F	GATGTAGGAGGGCGTGGATATGTCCT
HY/R	GTATTGACCGATTCCTTGCGGTCCGAA
H850F	TTCCTCCCTTTATTTCAGATTCAA
H852R	ATGTTGGCGACCTCGTATTGG
H855R	GCTGATCTGACCAGTTGC
H856F	GTCGATGCGACGCAATCGT
PKNTG-F	CTATAGGGCGAATTGGGTACCGACCTACCCATGTTTACTTG
PKNTG-R	GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCTGCTCTTG
GFP-R	GATGCCCTTCAGCTCGATGCGGTTCA

菌株 Strain	孢子浓度 Conidia concentration (×10⁶/m<BOLD>L</BOLD>)	孢子长度 Conidia length (μm)	隔膜数 Septa per conidium	菌丝融合率 Hyphal fusion rate (%)	孢子融合芽管率 CAT-mediated fusion rate (%)
WT	2.25±0.23	38.84±1.53	3.97±0.80	42.0±7.3	36.1±3.7
ΔFpAtg3	1.03±0.27**	23.72±2.03**	2.86±0.55**	22.2±5.4**	17.8±3.1**
FpAtg3-C	2.18±0.15	38.58±1.27	3.82±0.72	39.8±4.6	35.8±3.9