Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm²), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm²-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.     

胶孢炭疽菌附着胞形成过程中核相的动态变化

通过DAPI荧光染料染色观察胶孢炭疽菌Colletotrichumgloeosporioides附着胞发育过程中的核相动态变化,结果显示,第2次有丝分裂发生的部位在分生孢子产生芽管的一端中;分裂后,最接近芽管的一个子核移入芽管顶端,或通过芽管移入附着胞中。0.10μg/mL的三环唑可完全抑制附着胞中黑色素形成,但不影响核的分裂。三环唑处理12h后,发生2次有丝分裂数量约为73%,而发生3次有丝分裂的数量约为23.9%;绝大多数附着胞中是单核,双核数量小于5%。     

Regulation of pathogenic spore germination by CgRac1 in the fungal plant pathogen Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is a facultative plant pathogen: it can live as a saprophyte on dead organic matter or as a pathogen on a host plant. Different patterns of conidial germination have been recognized under saprophytic and pathogenic conditions, which also determine later development. Here we describe the role of CgRac1 in regulating pathogenic germination. The hallmark of pathogenic germination is unilateral formation of a single germ tube following the first cell division. However, transgenic strains expressing a constitutively active CgRac1 (CA-CgRac1) displayed simultaneous formation of two germ tubes, with nuclei continuing to divide in both cells after the first cell division. CA-CgRac1 also caused various other abnormalities, including difficulties in establishing and maintaining cell polarity, reduced conidial and hyphal adhesion, and formation of immature appressoria. Consequently, CA-CgRac1 isolates were completely nonpathogenic. Localization studies with cyan fluorescent protein (CFP)-CgRac1 fusion protein showed that the CgRac1 protein is abundant in conidia and in hyphal tips. Although the CFP signal was equally distributed in both cells of a germinating conidium, reactive oxygen species accumulated only in the cell that produced a germ tube, indicating that CgRac1 was active only in the germinating cell. Collectively, our results show that CgRac1 is a major regulator of asymmetric development and that it is involved in the regulation of both morphogenesis and nuclear division. Modification of CgRac1 activity disrupts the morphogenetic program and prevents fungal infection.     

组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响

以蛇足石杉Huperzia serrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因（histone methyltransferases,HMT）CgClr4和组蛋白去乙酰化酶基因（histone deacetylase,HDAC）CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR（RT-qPCR）检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzine A（HupA）产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。     

植物病原真菌1,8-间苯二酚黑色素研究进展

黑色素是一种广泛分布于生物体中的酚类聚合物疏水色素,分为1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素和3,4-二羟基苯丙氨酸(3,4-dihydroxyphenylalanine,L-DOPA)黑色素两种,其中DHN黑色素多存在于子囊菌门的植物病原真菌中。基因组和转录组技术的发展及功能基因组研究的深入,使DHN黑色素合成途径上关键基因在不同病原真菌中被鉴定,而且黑色素与真菌抗逆、发育和致病的关系受到越来越多的关注。本文阐述了DHN黑色素合成途径及其在真菌抗辐射与抗极端温度中的作用,以及黑色素对真菌侵染和细胞发育的影响,旨在加深人们对黑色素介导真菌与环境和寄主协同进化的认识,这对黑色素的基础研究和开发利用具有重要意义。     

The biosynthesis of melanin in Alternaria

A melanin which is insoluble in strong alkali has been isolated from Alternaria mycelium. Alkali fusion of the pigment produced p-hydroxybenzoa     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.     

Pathogenicity for onion and genetic diversity of isolates of the pathogenic fungus Colletotrichum gloeosporioides (Phyllachoraceae) from the State of Pernambuco, Brazil

Onion anthracnose, caused by Colletotrichum gloeosporioides, is one of the main diseases of onions in the State of Pernambuco. We examined the pathogenicity of 15 C. gloeosporioides strains and analyzed their genetic variability using RAPDs and internal transcribed spacers (ITS) of the rDNA region. Ten of the strains were obtained from substrates and hosts other than onion, including chayote (Sechium edule), guava (Psidium guajava), pomegranate (Punica granatum), water from the Capibaribe River, maracock (Passiflora sp), coconut (Cocus nucifera), surinam cherry (Eugenia uniflora), and marine soil; five isolates came from onions collected from four different regions of the State of Pernambuco and one region of the State of Amazonas. Pathogenicity tests were carried out using onion leaves and bulbs. All strains were capable of causing disease in leaves, causing a variable degree of lesions on the leaves; four strains caused the most severe damage. In the onion bulb tests, only three of the above strains caused lesions. Seven primers of arbitrary sequences were used in the RAPD analysis, generating polymorphic bands that allowed the separation of the strains into three distinct groups. The amplification products generated with the primers ITS1 and ITS4 also showed polymorphism when digested with three restriction enzymes, DraI, HaeIII and MspI. Only the latter two demonstrated genetic variations among the strains. These two types of molecular markers were able to differentiate the strain from the State of Amazonas from those of the State of Pernambuco. However, there was no relationship between groups of strains, based on molecular markers, and degree of pathogenicity for onion leaves and bulbs.     

Isolation and characterization of a clay-dispersing polysaccharide produced by the phytopathogenic fungus, Colletotrichum gloeosporioides

The fungus, Colletotrichum gloeosporioides, which is pathogenic to peppers produced an extracellular polysaccharide in liquid culture which possessed clay-dispersing activity. The polysaccharide could bind cationic dyes, Ruthenium Red and Alcian Blue, indicating it to be polyanionic. The polysaccharide dispersed kaolin in water and the dispersion was maintained for more than 7 days at 25 °C. Kaolin dispersion by the polysaccharide was stable from pH 3 to 10 but the addition of divalent metals at 1 mM inhibited half of the dispersion activity comparing to the control. The polysaccharide could disperse bentonite, calcium carbonate and other fine particles but did not possess emulsifying activity.     

Inhibitory effects of hinokitiol on tyrosinase activity and melanin biosynthesis and its antimicrobial activities

The inhibitory effects of hinokitiol, a constituent of the woody oils isolated from Cupressaceae heartwood, on mushroom tyrosinase and melanin formation in B16 melanoma cells as well as its antimicrobial activity were investigated. Our results showed that hinokitiol could strongly inhibit both monophenolase activity and diphenolase activity of the enzyme and the inhibition was reversible. The IC₅₀ values were estimated as 9.67?μM for monophenolase activity and 0.21?μM for diphenolase activity. The lag time of the monophenolase activity was not obviously lengthened by the compound. Kinetic analyses showed that the inhibition mechanism of hinokitiol was a mixed-type inhibition of the diphenolase activity. Hinokitiol effectively inhibited both cellular tyrosinase activity and melanin biosynthesis in B16 melanoma cells with significant cytotoxicity. Furthermore, it was found that hinokitiol could inhibit the proliferation of Salmonella enteritidis, Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, Klebsiella pneumoniae, and Ralstonia solanacearum to different extents. This research may widen the use of hinokitiol in the fields of food preservation, depigmentation, and insecticide use.     

Isolation of defense-related genes differentially expressed in the resistance interaction between pepper fruits and the anthracnose fungus Colletotrichum gloeosporioides

Unripe mature green fruits of pepper (Capsicum annuum) are susceptible to Colletotrichum gloeosporioides, whereas ripe red fruits are not. We established this pepper-C. gloeosporioides interaction as a model system to study the fungal resistance that develops during ripening of nonclimacteric fruit. Histochemical examination of transverse sections suggested that fungal invasion 24 h after inoculation (HAI) and colonization 48 HAI are critical events that differentiate between resistant and susceptible interactions. Based on this observation, we used messenger RNA differential display to isolate defense-related genes differentially expressed at 24 and 48 HAI. RNA gel blot analysis showed that six out of eighty cloned cDNAs were differentially expressed after infection of ripe fruit. The proteins encoded by these six clones, ddP1, ddP3, ddP4, ddP6, ddP13, and ddP47, showed significant homology to aldehyde dehydrogenase, P23 protein, NP24 protein, cytochrome P450 protein, esterase, and MADS-box protein, respectively, and may be involved in the resistance of ripe fruit to C. gloeosporioides infection.     

异齿裂腹鱼仔鱼的黑色素分布

本文运用组织学方法对异齿裂腹鱼仔鱼发育过程中除眼睛以外的黑色素分布进行研究.结果 表明:异齿裂腹鱼孵化出膜后,黑色素在各个组织器官的出现顺序为:脑颅外膜→围心腔、背部皮肤→腹腔内膜、脊髓腔.其中,出膜5d时脑颅外膜处出现黑色素;7d时围心腔和背部皮肤出现黑色素;10d时腹腔内膜、脊髓腔出现黑色素.异齿裂腹鱼出膜10 d...