苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能

【目的】研究表明,细胞色素P450(CYP)在死体营养型真菌的毒素合成代谢中发挥重要作用,预测可能与病原菌致病相关。论文对苹果树腐烂病菌(Valsa mali)毒素合成基因簇中的1个上调表达的CYP基因Vmcyp5进行生物学功能研究,明确CYP基因对病原菌致病力影响,为细胞色素P450基因家族对苹果树腐烂病菌致病机理的进一步研究提供依据。【方法】通过Double-joint PCR和PEG介导的原生质体转化技术获得具有G418抗性的突变体,并对突变体进行PCR检测及Southern blotting验证得到单拷贝敲除突变体。将目的基因片段重新导入敲除突变体,筛选获得互补突变体。最终对野生型菌株及敲除突变体、互补突变体进行菌落、产孢及致病力观察,利用SPSS软件对数据进行差异显著性分析,并利用q RT-PCR技术分析突变体黑色素基因簇的表达水平。【结果】通过基因敲除技术获得1个Vmcyp5基因的敲除突变体。与野生型菌株相比,Vmcyp5基因的敲除突变体菌落呈白色,产孢量减少51.3%。q RT-PCR分析发现敲除突变体黑色素基因簇基因表达量降低。重要的是,敲除突变体致病力较野生型菌株降低24.5%。互补突变体菌落颜色、产孢及致病力近似恢复至野生型菌株水平。【结论】Vmcyp5基因与病原菌黑色素合成、子实体的产生和致病力相关。     

The apple FERONIA receptor-like kinase MdMRLK2 negatively regulates Valsa canker resistance by suppressing defence responses and hypersensitive reaction

Valsa canker, caused by the fungus Valsa mali, is one of the most destructive diseases of apple trees in China and other East Asian countries. The plant receptor-like kinase FERONIA is involved in plant cell growth, development, and immunity. However, little is known about the function of FERONIA in apple defence against V. mali. In this study, we found that MdMRLK2 was highly induced by V. mali in twigs of V. mali-susceptible Malus mellana but not in those of the resistant species Malus yunnaensis. 35S:MdMRLK2 apple plants showed compromised resistance relative to wild-type (WT) plants. Further analyses indicated that 35S:MdMRLK2 apple plants had enhanced abscisic acid (ABA) levels and reduced salicylic acid (SA) levels relative to the WT on V. mali infection. MdMRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia-lyase (PAL), β-1,3-glucanase (GLU), and chitinase (CHT) during V. mali infection. Moreover, MdMRLK2 interacted with MdHIR1, a hypersensitive-induced response protein, and suppressed the MdHIR1-mediated hypersensitive reaction (HR), probably by impairing MdHIR1 self-interaction. Collectively, these findings demonstrate that overexpression of MdMRLK2 compromises Valsa canker resistance, probably by (a) altering ABA and SA levels, (b) suppressing polyphenol accumulation, (c) inhibiting PAL, GLU, and CHT activities, and (d) blocking MdHIR1-mediated HR by disrupting MdHIR1 self-interaction.     

Delimiting cryptic pathogen species causing apple Valsa canker with multilocus data

Fungal diseases are posing tremendous threats to global economy and food safety. Among them, Valsa canker, caused by fungi of Valsa and their Cytospora anamorphs, has been a serious threat to fruit and forest trees and is one of the most destructive diseases of apple in East Asia, particularly. Accurate and robust delimitation of pathogen species is not only essential for the development of effective disease control programs, but also will advance our understanding of the emergence of plant diseases. However, species delimitation is especially difficult in Valsa because of the high variability of morphological traits and in many cases the lack of the teleomorph. In this study, we delimitated species boundary for pathogens causing apple Valsa canker with a multifaceted approach. Based on three independent loci, the internal transcribed spacer (ITS), β‐tubulin (Btu), and translation elongation factor‐1 alpha (EF1α), we inferred gene trees with both maximum likelihood and Bayesian methods, estimated species tree with Bayesian multispecies coalescent approaches, and validated species tree with Bayesian species delimitation. Through divergence time estimation and ancestral host reconstruction, we tested the possible underlying mechanisms for fungal speciation and host‐range change. Our results proved that two varieties of the former morphological species V. mali represented two distinct species, V. mali and V. pyri, which diverged about 5 million years ago, much later than the divergence of their preferred hosts, excluding a scenario of fungi–host co‐speciation. The marked different thermal preferences and contrasting pathogenicity in cross‐inoculation suggest ecological divergences between the two species. Apple was the most likely ancestral host for both V. mali and V. pyri. Host‐range expansion led to the occurrence of V. pyri on both pear and apple. Our results also represent an example in which ITS data might underestimate species diversity.     

苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能

【目的】非核糖体多肽合成酶(NRPS)在植物病原真菌与其寄主互作过程中发挥着重要作用,明确Vm NRPS12基因在苹果树腐烂病菌致病过程中的功能,将为今后深入研究苹果树腐烂病菌NRPS作用机制提供理论依据。【方法】基于苹果树腐烂病菌全基因组数据,得到VmNRPS12基因。运用qRT-PCR技术分析VmNRPS12在侵染初期的表达水平,利用Double-joint PCR和PEG介导的原生质体转化获得该基因抗潮霉素的突变体,对突变体进行PCR检测及Southern blot验证得到敲除突变体,进一步通过重新导入该基因全长片段获得互补突变体,最后对野生型、敲除突变体和互补突变体进行菌落、产孢及致病力观察,对检测数据用SPSS软件进行差异显著性分析。【结果】定量分析显示该基因在侵染初期显著上调表达,且接种48 h后的表达量是对照的138.6倍。该基因的敲除突变体在营养生长及产孢方面与野生型菌株03-8相比无显著性差异,但致病力与野生型菌株03-8相比显著减弱,且互补突变体致病力近似恢复至野生型水平。【结论】VmNRPS12基因与苹果树腐烂病菌致病性相关。     

Vm-milR21调控苹果黑腐皮壳侵染致病的功能和机理

苹果黑腐皮壳(Valsamali)引起的腐烂病是苹果最具毁灭性的枝干病害。微小核糖核酸(microRNA-like RNAs, milRNAs)在真菌生长发育、侵染致病和胁迫响应等过程中发挥重要作用。前期研究发现Vm-milR21在病菌侵染阶段特异性下调表达,推测其可能参与V. mali的侵染致病过程。【目的】本文对Vm-milR21的功能进行研究。【方法】制备Vm-milR21前体过表达和沉默转化株,评价不同转化株与野生型菌株表型差异。进而,通过实时荧光定量聚合酶链反应(quantitative real time polymerase chain reaction, qRT-PCR)和共转化技术验证Vm-milR21与其潜在靶标Vm-03494之间的靶向调控关系。在此基础上,构建Vm-03494的基因敲除突变体,并鉴定突变体表型。【结果】与野生型菌株相比,过表达转化株的菌丝生长速率以及对叶片和枝条的致病力均大幅降低,而沉默转化株无明显变化。Vm-milR21能够序列特异性地抑制Vm-03494表达。相较于野生型菌株,Vm-03494敲除突变体菌丝生长速率和致病力均显著降低。【结论】...     

Hce2 domain-containing effectors contribute to the full virulence of Valsa mali in a redundant manner

Valsa mali is the causal agent of apple Valsa canker, a destructive disease in East Asia. Effector proteins play important roles in the virulence of phytopathogenic fungi, and we identified five Hce2 domain-containing effectors (VmHEP1, VmHEP2, VmHEP3, VmHEP4 and VmHEP5) from the V. mali genome. Amongst these, VmHEP1 and VmHEP2 were found to be up-regulated during the early infection stage and VmHEP1 was also identified as a cell death inducer through its transient expression in Nicotiana benthamiana. Although the deletion of each single VmHEP gene did not lead to a reduction in virulence, the double-deletion of VmHEP1 and VmHEP2 notably attenuated V. mali virulence in both apple twigs and leaves. An evolutionary analysis revealed that VmHEP1 and VmHEP2 are two paralogues, under purifying selection. VmHEP1 and VmHEP2 are located next to each other on chromosome 11 as tandem genes with only a 604 bp physical distance. Interestingly, the deletion of VmHEP1 promoted the expression of VmHEP2 and, vice versa, the deletion of VmHEP2 promoted the expression of VmHEP1. The present results provide insights into the functions of Hce2 domain-containing effectors acting as virulence factors of V. mali, and provide a new perspective regarding the contribution of tandem genes to the virulence of phytopathogenic fungi.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

拮抗植物病原真菌链霉菌菌株ZH-356的鉴定及其生防评价

[目的]对实验室分离到的菌株ZH-356进行鉴定并评价其对植物病原真菌的生物防治效果,为研发针对植物真菌病害的生防菌剂提供理论指导。[方法]通过平板对峙法确定菌株ZH-356抗菌谱,并通过16S rRNA基因序列分析确定其种属,利用离体枝条的苹果树腐烂病菌感染预防试验和患腐烂病苹果树的防治试验评价其生防效果。[结果]菌株ZH-356鉴定为链霉菌属,与直丝紫链霉菌(Streptomyces rectiviolaceus)相似性最高,为99.71%。抗菌谱试验表明,菌株ZH-356对苹果树腐烂病菌、小麦赤霉病菌、小麦根腐病菌和番茄早疫病菌等多种植物病原真菌均具有较强的抑制作用,这种抑制作用可导致苹果树腐烂病菌菌丝变粗、交叉扭曲、分支变少且容易断裂。此外,ZH-356产生的抑菌活性物质对温度和酸碱度具有高度稳定性,并且该活性物质只存在于其胞内,只有当ZH-356遇到植物病原真菌时才会被分泌出来以抑制它们的生长。在离体枝条的苹果树腐烂病菌感染预防试验中,ZH-356对苹果树腐烂病防效可达94%以上,而在患腐烂病苹果树的防治试验中,ZH-356菌制剂对苹果树腐烂病的防效高达100%。[结论]链霉菌ZH-356抑菌谱广,对多种植物病原真菌均具有良好的拮抗活性,可作为防治植物真菌病害的生防菌株,为基于ZH-356菌株的生防菌剂的开发和防治苹果树腐烂病等植物真菌病害奠定了基础。     

Screening and biological function of protein elicitor secreted from biocontrol agent Hhs.015

Strain Hhs.015, a PGPR strain well‐studied for biocontrol of apple Valsa canker, was isolated from cucumber roots and classified as a new species of Saccharothrix yanglingensis. Hhs.015 was able to colonize apple tissue culture seedlings and trigger disease resistance. According to the whole‐genome sequence of S. yanglingensis Hhs.015, its secreted proteins were predicted and analysed, which can help to find out Hhs.015 functional substances that are involved in inducing plant system resistance and also the molecular mechanisms of the interaction between Hhs.015 and the host plants. Using the software SignalP4.1, TMHMM2.0, DAS‐TMfilter, HMMTOP, ScanProsite, PSORT and big‐PI predictor, 7,379 ORFs of the genome of S. yanglingensis Hhs.015 were predicted and analysed. Moreover, the function of ORFs where the signal peptides come from was predicted by Swissprot database. The selected proteins were obtained by prokaryotic expression. Their effects in inducing plant resistance to the pathogen Valsa mali, resistance‐related enzyme activity and growth promotion analysis were evaluated. In total, 158 ORFs with known function, such as serine proteinase and transpeptidase, were identified; the remaining 142 ORFs were function‐unknown putative proteins. Five putative proteins were successfully obtained by prokaryotic expression, and the results showed that the protein named 5620g could enhance the plant disease resistance to the pathogen V. mali, inhibit the expansion of lesions and upregulate the defence‐related enzyme activity. The protein named 3176g could prolongate cucumber root. In conclusion, biocontrol strain Hhs.015 may secrete protein elicitors to improve plant resistance to the pathogenic microorganism and promote the growth of host plants.     

Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096

Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE‐1, AtFAE‐2, and AtFAE‐3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH‐dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5–8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca²⁺, K⁺, and Mg²⁺ stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k_cat/K_m) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF‐MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE‐1 belonged to type A while AtFAE‐2 and AtFAE‐3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:924–932, 2013     

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Growth inhibition effect of pyroligneous acid on pathogenic fungus,<Emphasis Type="Italic">Alternaria mali</Emphasis>, the agent of Alternaria blotch of apple

We investigated the growth inhibition effect of pyroligneous acid on the pathogenic fungus,Alternaria mali, which is known to be the agent of Alternaria blotch of apple plants. Chemical control ofA. mali could be achieved through the use of agrochemical fungicides, while the substitute for agrochemical control is gradually increasing. It was observed that pyroligneous acid exhibited antifungal activity against some plant pathogenic fungi. More specifically, the growth ofA. mali was completely inhibited in pyroligneous acid at a dilution of 1∶32. When its antifungal activity was compared to that of polyoxin B, which is used for the chemical control of Alternaria blotch of apple, it was observed that the antifungal activity of pyroligneous acid diluted at 1∶32 corresponded to 2.0 mg/mL of polyoxin B. Consequently, it is concluded that the diluted pyroligneous acid can substitute for polyoxin B, thereby reducing the use of the agrochemical for the control of Alternaria blotch of apple.     

新疆野果林苹果腐烂病病原菌鉴定及药用植物内生细菌对其抑菌效果

【背景】药用植物内生细菌能产生与寄主植物相同或相似的化合物及一些新的次级代谢产物等,具有促进宿主植物生长、抵抗病虫害、降解有毒有害化合物等作用。【目的】进一步提高苹果腐烂病生物防治的效率,丰富新疆药用植物内生细菌拮抗功能菌株的资源库。【方法】从新疆伊犁新源县和塔城额敏县野果林中采集带腐烂病病斑的果树枝条,分离鉴定苹果腐烂病病原菌,并采用平板对峙法从药用植物内生细菌中筛选对苹果腐烂病具有抑制作用的拮抗菌株。【结果】从两地共分离获得234株分离株,筛选鉴定出25株Valsa malicola和2株Valsa mali;同时,筛选出92株具有抑菌效果的内生细菌菌株,其中70株来自甘草植物内生细菌。【结论】药用植物甘草中富含较为丰富的抗苹果腐烂病病原菌的微生物菌株资源。本研究在新疆野果林苹果腐烂病的生物防治及药用植物内生细菌的开发利用等方面具有重要意义。     

The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens

Plant‐pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F‐box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root‐infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant‐pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F‐box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.     

Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k_cat/K_m)]_CE/[(k_cat/K_m)]_FAE factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys²⁸¹, Asp²⁸², Asn³¹⁶ and Lys³¹⁷) situated close to the catalytic core (Ser¹⁴³/Asp²⁷³/His³⁰⁵) and a deletion of a 34-AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k_cat/K_m values) and enzymes with inverted specificity ((k_cat/K_m)_CE/(k_cat/K_m)_FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol⁻¹), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol⁻¹) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.