On the Safety of Aspergillus Niger – a Review

• Loading metrics

Open Admission

Peer-reviewed

Inquiry Commodity

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

• Cheng-Qian Liu,
• Kang-Di Hu,
• Ting-Ting Li,
• Ying Yang,
• Feng Yang,
• Yan-Hong Li,
• He-Ping Liu,
• Xiao-Yan Chen,
• Hua Zhang

x

• Published: March three, 2017
• https://doi.org/10.1371/journal.pone.0173277

Figures

Abstruse

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade institute cell wall. To evaluate whether the deletion of an exo-polygalacturonase cistron pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.fifteen (wild type) via homologous recombination. The ΔpgxB mutant showed like growth beliefs compared with the wild type. Pectin medium induced pregnant college expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. All the same, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused past ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, cistron expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results testify that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple tree fruit, suggesting that pgxB plays an important role in the infection process on the apple tree fruit.

Commendation: Liu C-Q, Hu K-D, Li T-T, Yang Y, Yang F, Li Y-H, et al. (2017) Polygalacturonase cistron pgxB in Aspergillus niger is a virulence factor in apple fruit. PLoS ONE 12(3): e0173277. https://doi.org/x.1371/periodical.pone.0173277

Editor: Sabrina Sarrocco, Universita degli Studi di Pisa, ITALY

Received: October 29, 2016; Accepted: February 17, 2017; Published: March 3, 2017

Copyright: © 2017 Liu et al. This is an open admission article distributed under the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in whatsoever medium, provided the original writer and source are credited.

Information Availability: All relevant data are within the paper.

Funding: The funders National Natural Scientific discipline Foundation of Cathay, Anhui Provincial Science and Applied science Major Project, and Anhui Provincial Education Section had no part in written report design, data collection and assay, conclusion to publish, or preparation of the manuscript. Anhui Siping Food Development Co. Ltd. provided support in the course of salaries for authors YHL and HPL, but did non take any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' department.

Competing interests: We accept the following interests. Yan-Hong Li and He-Ping Liu are employed by Anhui Siping Food Development Co. Ltd. There are no patents, products in development or marketed products to declare. This does not modify our adherence to all the PLOS ONE policies on sharing data and materials, every bit detailed online in the guide for authors.

Introduction

Pectinases are the most important pathogenic cistron in plant pathogenic bacteria and fungi [1–iv]. They are responsible for pathogens to decompose pectin in plant prison cell wall. Pectin hydrolysis not just weakens the cell wall to facilitate penetration and colonization of the host, it also provides the fungus carbon sources for its growth [v]. Pectinases are consisted of polygalacturonase (PG), pectin lyase (PNL), pectate lyase (PL), pectinesterase (PE), pectin methyl esterase (PME). Polygalacturonase is i of the major members of pectinases which cleaves α-i,4-glycosidic of D-galacturonic acid in pectin and it is classified into endo- and exo-polygalacturonase on the basis of the way of eliminating galacturonic acid [6,7].

The production of PG by pathogenic fungi is critical for their success and survival during host infection [8]. Information technology has been confirmed that the loss of a polygalacturonase gene in some fungi would issue in decreased pathogenicity. Shieh et al [9] showed that a polygalacturonase gene is related to the infection of Aspergillus flavus in cotton bolls. The disruption of endo-polygalacturonase factor Bcpg1 or Bcpg2 in Botrytis cinerea reduced its virulence on different hosts [ten]. It is also reported that the loss of pectin methyl esterase factor Bcpme1 reduced virulence on apples, and pectin lyase pelB was an important virulence factor in Colletotrichum gloeosporioides when attacking avocado [11,12]. PG is also required for infection past Phytophthora capsici and Alternaria citri [thirteen,fourteen]. Nonetheless there are also studies demonstrated that disruption of some polygalacturonase genes in fungi did not directly affect virulence, for instance, deletion of PG1, PG5, PGX4 in Fusarium oxysporum led to no virulence deviation in tomato [15,16] and endopolygalacturonase PGN1 is not required for pathogenicity of Cochliobolus carbonum on maize [17]. Mutants lacking both polygalacturonase genes cppg1/cppg2 in Claviceps purpurea did not affected vegetative properties, just they are nearly nonpathogenic on rye [18].

Aspergillus niger is a saprophytic mucus which degrades plant cell wall polysaccharides and leads to the disuse of fruits and vegetables [nineteen]. Since the 1990s, technical advances in molecular biological science speed up the functioning mechanism of A. niger and A. niger gene sequencing has been completed [20,21]. Deletion of kusA factor in A. niger dramatically improved homologous integration efficiency and facilitated gene knockout in A. niger [22]. However, whether polygalacturonase contributes to the pathogenicity of A. niger on fruit is still unclear. Here we constructed pgxB deletion mutant in A. niger MA lxx.15 via homologous recombination and the pathogenicity were evaluated in ΔpgxB strain.

Textile and methods

Fungal strains and growth atmospheric condition

A. niger MA 70.xv (ΔpyrG, pyrG encodes orotidine-v-phosphate decarboxylase, cell lacking this enzyme cannot grow without exogenous uridine, but can resist toxicity of 5-Fluoroorotic acid) was used as wild blazon strain in this piece of work. A. niger was grown on potato dextrose agar medium (PDA) (per liter: 200 g of potato; twenty g of agar; xx 1000 of glucose and 10 mM uridine) at 28°C. Therefore all medium used in this report were supplemented with 10 mM uridine except where stated. For in vitro growth evaluation, A. niger spores were suspended in sterile water and adjusted to x⁶ spores per mL. To test whether the absence of pgxB afflicted mycelial growth, PDA and pectolytic enzyme-inducing medium (PEIM) (per liter: 20 g of agar; xx g of pectin; MM medium (5 chiliad of KNO_iii; 0.3 g of KCl; 2 k of MgSO₄·7H₂O; 5 g of KH₂PO_iv; 0.008 mg of Na_twoB₄O₇·10H₂O; 0.sixteen mg CuSO₄·5H_twoO; 0.256 mg of FeCl₃·6H_twoO; 0.1213 mg of MnSO₄·4H₂O; 0.16 mg of NaMoO₄·2H₂O; 2.85 mg of ZnSO₄) plus 10 mM uridine) [23] were spot-inoculated with v μL spore interruption of wild blazon and ΔpgxB mutant. All strains were inoculated onto iii plates of each medium and colony bore was measured daily.

For the decision of growth curve, 1 mL spore intermission of wild type and ΔpgxB mutant were inoculated in 100 mL Erlenmeyer flasks containing 30 mL spud dextrose medium or PEIM at thirty°C, 150 rpm. Mycelium was harvested on a quantitative filter paper at the time of 12, 24, 36, 48, 60, 72, 84, 96 h and weighed.

Vector construction and transformation

The exo-polygalacturonase factor pgxB in A. niger was deleted following gene knockout method of Delmas et al [24]. Upstream and downstream Dna fragments AB (548 bp) and CD (564 bp) flanking pgxB factor were amplified by polymerase chain reaction (PCR) from A. niger MA seventy.fifteen genomic DNA. Primers were designed using the genome database A. niger CBS 513.88 and the upstream and downstream fragments independent a common HindIII restriction site to ligate them together and EcoRI and XhoI restriction sites were used for cloning the joined fragment into the plasmid pC3 [24] to create pC3-An_ΔpgxB integrative plasmid. Primers are shown in Tabular array 1.

A. niger protoplast preparation and transformation were carried out by the method of Baltz et al [23]. 4-day-sometime mycelia grown from PDA were harvested and were digested with 0.four g Lyticase (Sigma) to obtain fungal protoplasts. Protoplasts were transformed with x μg pC3-An_ΔpgxB (un-linearized) in l μL polyethylene glycol 6000. Transformations were inoculated on upper layer of transformation medium without uridine (per liter: upper medium: MM; six thou of agar; 0.95 M sucrose; lower medium: MM; 12 g of agar; 0.95 M sucrose) to select for the integration of the plasmid carrying pyrG on the chromosome. Transformants were purified past breeding them twice successively on the aforementioned transformation medium but lacking sorbitol (per liter: MM, 20 g of agar). Transformants were then propagated twice on PDA medium containing 10 mM uridine to release the selective pressure level on the integrated plasmid. For selecting clones that had excised the plasmid (ΔpyrG), spores were then spread on MM-Uri-5-FOA medium (per liter: MM; 20 grand of agar; 10 grand of glucose; 1.6 mM uridine; 750 μg/mL five-fluoro-orotic acid). Clones from last medium were cultured in MM-Uri-5-FOA liquid medium at 250 rpm at xxx°C for three d, and mycelia were harvested for genomic DNA extraction.

DNA extraction and PCR confirmation of ΔpgxB strain

Genomic DNA was extracted using Master Pure Yeast DNA Purification Kit (Epicentre). Primers pgxB-A, pgxB-D and another primers internal to the pgxB factor pgxB-E1, pgxB-E2 (PCR product size: 953 bp) were designed using Primer Premier 5.0 software. Primers are shown in Table 1.

Determination of PG activity

For determination of polygalacturonase activity, 100 μL of conidia (one×10⁶ spores per mL) were inoculated into a 100 mL Erlenmeyer flask containing 30 mL of liquid PEIM and cultured at 30°C for 5 days. Civilisation medium after suction filtration was used for PG activity assay post-obit the method described past Miller [25]. Reaction mixture was consisted of 1 mL of l mM acetic acid-sodium acetate at pH5.five, 0.five mL of 10 g/L pectin solution and 0.five mL of crude enzyme or enzyme boiled for 5 min followed by incubation at 40°C for thirty min. Reactions were terminated by calculation 1.5 mL of DNS (3,5-dinitrosalicylic acrid) followed past a v min incubation in a boiling h2o bath. The reaction mixture was cooled to room temperature, and distilled water was added to a concluding book of 25 mL. Absorbance at 540 nm was measured. One unit of PG was defined every bit 1 μg of galacturonic acrid produced by pectin per hour and expressed as U/mL enzyme excerpt.

PG activity was also determined by plate analysis. The wild type and ΔpgxB mutant were inoculated on PEIM and cultured for 3 days at xxx°C. Thereafter the colonies were rinsed off the plates with distilled water before staining the plates with 0.05% ruthenium ruby. Pectinase production was evaluated past ratio of diameter of clear zone formed around colonies relative to diameter of mycelia [26,27].

Virulence assay of A. niger on apple fruit

Apple fruits were washed with tap water and and so surface-sterilized with 75% ethanol. Five dissimilar sites on the surface of apples were wounded (2 mm diameter and 5 mm deep) and injected with v μL wild type or ΔpgxB mutant spore suspension (10^{half dozen} spores per mL) respectively. After air-drying, iii replicates were put in sealed container with H_twoO at the bottom at 25°C. The necrosis bore were measured daily after inoculation. Spores grown from wounds were used for RNA extraction and quantitative PCR.

RNA extraction and quantitative PCR

RNA was extracted from frozen mycelium grown on PDA, PEIM or apples ground in liquid nitrogen with TRNzol RNA Reagent kit (Tiangen). After DNase handling, cDNA were obtained according to the manufacturer's instruction of PrimeScript RT Main Mix (TaKaRa, Japan). Primers used for quantitative PCR are shown in Table 1. Relative quantification was processed using the method of Delta-Ct.

Statistical analysis

Statistical significance was tested by i-way assay of variance (ANOVA), and the results are expressed equally the hateful values ± standard deviation (SD) of three independent experiments. Fisher's least significant differences (LSD) were calculated post-obit a meaning (P < 0.01 or P < 0.05) t exam.

Results

Gene disruption of pgxB in A. niger

548-bp upstream and 564-bp downstream Dna fragments AB and CD were amplified by PCR from A. niger MA 70.fifteen genomic Dna. Fragment ABCD (1094 bp) past the ligation of AB and CD were cloned into plasmid pC3 to generate the recombinant plasmid pC3-An_ΔpgxB (6959 bp).

Then the plasmid pC3-An_ΔpgxB was transformed and integrated into A. niger MA seventy.xv protoplast and the ΔpgxB mutant was confirmed past genomic PCR. As shown in Fig 1, genomic Deoxyribonucleic acid was used equally template for PCR confirmation with primers pgxB-A and pgxB-D which flank pgxB gene and primers on the ORF of pgxB gene pgxB-E1 and pgxB-E2. 1100 bp band for ΔpgxB and 3500 bp for the wild type were obtained by the primers pgxB-A and pgxB-D, and a 953-bp band for the wild type and no amplification for ΔpgxB with the primers pgxB-E1 and pgxB-E2. All of these results confirmed that the pgxB gene was disrupted in ΔpgxB.

Fig i. Confirmation of pgxB deletion mutant by PCR analysis of genomic DNA from wild type and ΔpgxB.

Lane 1 and 2, genomic PCR of A. niger MA seventy.15 and ΔpgxB with external primers respectively; lane 3 and 4, genomic PCR of A. niger MA seventy.fifteen and ΔpgxB with internal primers respectively.

https://doi.org/x.1371/journal.pone.0173277.g001

Growth analysis of ΔpgxB mutant strain

Pectinases in some fungi can be induced past pectin [28]. To evaluate whether the lack of pgxB would affect its growth on pectin medium, we compared growth of ΔpgxB and A. niger MA seventy.fifteen on PDA (no pectin) and pectin medium (PEIM). The radial growth was measured on solid media. Information technology was found that growth rate, estimated equally colony diameter, showed no difference between the ΔpgxB mutant and wild type on PDA (Fig 2A and 2B) or pectin medium (Fig 2C and 2d). Besides, no significant difference in growth bend was constitute between ΔpgxB mutant and the wild type in liquid medium with or without pectin (Fig 2E and 2F).

Fig 2. Radial mycelia growth of ΔpgxB strain on PDA and PEIM medium.

A and B show mycelia growth and bore analysis of the wild type A. niger MA 70.xv (WT) and ΔpgxB mutant on PDA for 96 h; C and D prove mycelia growth and mycelia bore of the wild blazon A. niger MA lxx.15 (WT) and ΔpgxB mutant on PEIM for 108 h. Eastward and F, dry weight of the wild type A. niger MA 70.15 (WT) and ΔpgxB strain in potato dextrose and pectolytic enzyme-inducing liquid medium for 96 h.

https://doi.org/10.1371/journal.pone.0173277.g002

Inducing effect of pectin on the expression of pectinase genes in A. niger

To report whether the expression of pectinase genes were induced by pectin, relative expression of different pectinase genes in ΔpgxB and A. niger MA 70.xv on PDA and pectin medium (PEIM) were determined by quantitative PCR. As shown in Fig 3A, expression of pgxB cistron in wild type was significantly enhanced by pectin medium, while the expression was not detected in ΔpgxB, farther confirming that pgxB cistron was deleted in the mutant. For wild blazon, most of pectinase genes similar PG genes pgaII, pgaB, pgaD, pgaE, pgaX, pgxA, pgxC, PL genes pelA, pelC, pelD, pelF, plyA, PME cistron An04g09690, PE gene An02g12505 showed enhanced expression on PEIM than those on PDA, suggesting that the expression of pectinases genes were induced by pectin (Fig 3B and 3C). Similarly, enhanced gene expression of PG genes pgaII, pgaA, pgaB, pgaD, pgaE, pgaX, pgxA, pgxC, PL genes pelA, pelC, pelD, pelF, plyA, PME gene An04g09690, PE gene An02g12505 was also observed in ΔpgxB mutant (Fig 3D and 3E).

Fig 3. Pectin medium (PEIM) induces college expression of pectinase genes in wild type and ΔpgxB mutant grown on PDA and PEIM.

A, relative expression of pgxB in A. niger MA lxx.15 and ΔpgxB mutant on PDA and PEIM; B and C, relative expression of pgaI, pgaII, pgaA, pgaB, pgaC, pgaD, pgaE, pgaX, pgxA, pgxC, pelA, pelB, pelC, pelD, pelF, pmeA, An04g09690, plyA and An02g12505 in A. niger MA 70.15 on PDA and PEIM medium respectively for 4 days; C and D, relative expression of pgaI, pgaII, pgaA, pgaB, pgaC, pgaD, pgaE, pgaX, pgxA, pgxC, pelA, pelB, pelC, pelD, pelF, pmeA, An04g09690, plyA and An02g12505 in ΔpgxB mutant on PDA and PEIM medium respectively for 4 daysouthward. * and ** in this figure and following ones stand up for a meaning difference between two data at P < 0.05 and P < 0.01, respectively.

https://doi.org/x.1371/periodical.pone.0173277.g003

Polygalacturonase activeness analysis

pgxB gene is a fellow member of polygalacturonase family plant in A. niger. To report whether pgxB gene actually contributes to the whole activity of polygalacturonase in A. niger, polygalacturonase activity was determined in ΔpgxB mutant. The ΔpgxB mutant and A. niger MA 70.15 were grown in liquid PEIM for 3 days and the activity of secreted polygalacturonase in the medium was analyzed. As shown in Fig 4A, ΔpgxB exhibited lower PG activity (by 5.eight%) compared with the wild type. Secreted PG activity by A. niger was also assayed on plate. As shown in Fig 4B and 4C, the ratio of diameter of clear zone (Dc) which showing the deposition of pectin past secreted PG and mycelia (Dm) produced by ΔpgxB was smaller (by 6.v%) than that of wild blazon, indicating that ΔpgxB mutant produced less pectinase than the wild type A. niger MA 70.15.

Fig 4. Activity evaluation of secreted polygalacturonase (PG) from A. niger MA 70.15 and ΔpgxB mutant.

A, the wild blazon A. niger MA lxx.15 (WT) and ΔpgxB strains were grown in liquid pectolytic enzyme-inducing media (PEIM) for 5 days and 0.five mL supernatant of PEIM cultures was sampled for polygalacturonase activity assay. B, The wild blazon and ΔpgxB mutant were inoculated on pectolytic enzyme-inducing media (PEIM) and cultured for 3 days at xxx°C and the plates were stained with 0.05% ruthenium red and photographed. C, ratio of bore of action zone (articulate zone, Dc) relative to the diameter of mycelia (Dm).

https://doi.org/ten.1371/journal.pone.0173277.g004

Pathogenicity assay on apple tree fruit

Whether pectinase was a pathogenic cistron in A. niger infection on fruit is yet unknown, thus nosotros studied the virulence of ΔpgxB mutant on fruit. Apple tree fruits were inoculated with conidial suspension of ΔpgxB and the wild type A. niger MA lxx.fifteen. Lesion evolution was monitored daily and the bore was measured. As shown in Fig 5A and 5B, pgxB disruption resulted in a reduction of decay development every bit the lesion diameter caused by ΔpgxB was about twenty% smaller than that of wild type on 4, 5, 6, 7 days and 15% smaller on 10, eleven days, suggesting that the virulence of ΔpgxB on apple fruit was significantly lower than that of wild blazon.

Fig five. Pathogenicity assay on apples with the wild type and ΔpgxB mutant.

A and B, the wild type A. niger MA 70.fifteen and ΔpgxB mutant grown on apples at 4, 5, 6, 7, 10 and 11 days post-inoculation and the lesion diameter acquired by wild type and ΔpgxB are shown. C and D, relative expression of unlike pectinase genes in A. niger MA seventy.15 (WT) and ΔpgxB mutant after infecting apples for xvi days.

https://doi.org/10.1371/journal.pone.0173277.g005

To written report why the virulence was decreased in ΔpgxB, expression of various pectinase genes in ΔpgxB and wild blazon in the process of infecting apples was adamant past quantitative PCR. Expression of PG genes pgaI, pgaII, pgaA, pgaC, pgaD, pgaE, pgaX in ΔpgxB were higher in ΔpgxB than those in wild type, while there was no significant difference in expression of PG genes pgaB, pgxA, pgxC, PL genes pelA, pelB, pelC, pelD, pelF, plyA and PME genes pmeA, An04g09690 betwixt ΔpgxB mutant and wild type (Fig 5C and 5D). The increased expression of PG genes in ΔpgxB suggested a possible compensation result in pgxB deletion mutant.

Give-and-take

Four exo-polygalacturonase genes were plant in A. niger, including pgxA, pgxB, pgxC and pgaX [29]. Besides, endo-polygalacturonase gene pgaI, pgaII, pgaA, pgaB, pgaC, pgaD, pgaE, pectin lyase gene pelA, pelB, pelC, pelF, plyA, pectin methylesterase factor pmeA, An04g09690 accept been isolated and sequenced from A. niger [xxx–36]. The factor pgxB in A. niger (cistron ID: 4980661), consisting of 439 amino acids, with five exons and 4 introns, a molecular mass of 67 kDa, encodes the extracellular exo-polygalacturonase and it was studied in the nowadays inquiry.

In this newspaper, we described the construction of a mutant disrupted in the pgxB gene in A. niger. ΔpgxB exhibited no growth rate reduction on PDA and pectin medium compared with wild type, which means the disruption of pgxB did not weaken its ability of decomposing pectin as carbon source (Fig two). In contrast, Bcpme1 mutant in B. cinerea and pelB mutant in Colletotrichum gloeosporioides showed reduced growth on pectin medium [11,12]. Pectinase was induced by pectin, polygalacturonic acid or galacturonic acid and was repressed by glucose and polygalacturonase-inhibiting protein (PGIP) [37,38]. Quantitative PCR results showed that expression of near of pectinase genes such as PG genes pgaII, pgaA, pgaB, pgaD, pgaE, pgaX, pgxA, pgxC, PL genes pelA, pelC, pelD, pelF, plyA were up-regulated on pectin medium compared that on PDA (Fig 3), confirming that the expression of pectinase genes were induced by pectin. Nosotros as well found that ΔpgxB mutant grown in liquid and solid PEIM showed significant lower PG activity than the wild-blazon strain, suggesting that the loss of pgxB reduced the production of PG (Fig iv).

Virulence analysis on apple fruit showed that the deletion of pgxB factor has a profound effect on lesion development in the infection of apple as lesion diameter caused past ΔpgxB was smaller than that of wild blazon. A similar reduction in maceration power has been observed with pectinase-deficient mutants of phytopathogenic bacteria such equally Erwinia, Pseudomonas and Ralstonia species [39,twoscore]. Besides, Oeser et al. [18] plant that mutant in both cppg1 and cppg2 are almost non-pathogenic on rye using a factor-replacement arroyo. However, pectinases are usually encoded by multiple genes, thus mutation in 1 pectinase factor might non affect the virulence on fruits [15–17]. The disruption of either the pelA or pelD cistron in Nectria hematococca alone causes no detectable decrease in virulence, whereas disruption of both pelA and pelD drastically reduces virulence [41]. In social club to empathise the decreasing virulence of ΔpgxB mutant, expression of some pectinase genes were assayed, and nosotros found that PG genes pgaI, pgaII, pgaA, pgaC, pgaD, pgaE and pgaX were expressed moore highly in ΔpgxB mutant than in the wild blazon. Deletion of one gene in a gene family might result in higher expression of other genes with the same part every bit polygalacturonases of A. niger are encoded past a family unit of diverged genes [42]. A like phenomenon that disruption of serine proteinase acquired an increment in metalloproteinase has also been found in Aspergillus flavus [43]. Withal, the lack of pgxB all the same dramatically reduced lesion bore on apples. Our results demonstrate that pgxB is a virulence factor which partially contributes to the virulence of A. niger on apple fruit, thus highlighting the demand for farther research to elucidate the roles of other pectinase genes in A. niger.

Acknowledgments

We are grateful to Arthur F. J. Ram at Academy of Leiden for providing the strain Aspergillus niger MA 70.15 and David B. Archer at University of Nottingham for providing the plasmid pC3.

Author Contributions

1. Conceptualization: CQL KDH TTL HZ.
2. Data curation: CQL KDH YY FY HZ.
3. Formal analysis: CQL TTL YHL HPL.
4. Funding acquisition: KDH YY HZ.
5. Investigation: CQL KDH TTL YY.
6. Methodology: CQL YY FY YHL.
7. Projection administration: KDH YHL HPL HZ.
8. Resources: YY FY YHL HPL HZ.
9. Software: CQL KDH HPL HZ.
10. Supervision: HPL HZ.
11. Validation: CQL KDH XYC HZ.
12. Visualization: CQL KDH HZ.
13. Writing – original draft: CQL KDH HZ.
14. Writing – review & editing: CQL KDH TTL XYC HZ.

References

1. 1. Herron SR, Benen JA, Scavetta RD, Visser J, Jurnak F. Structure and function of pectic enzymes: virulence factors of constitute pathogens. Proceedings of the National Academy of Sciences. 2000; 97(xvi): 8762–8769.
   • View Article
   • Google Scholar
2. 2. Accept AT, Tenberge KB, Benen JAE, Tudzynski P, Visser J, van Kan JAL. The contribution of prison cell wall degrading enzymes to pathogenesis of fungal plant pathogens. Springer Berlin Heidelberg. 2002; 341–358 p.
3. 3. Schmalhorst PS, Krappmann S, Vervecken West, Rohde One thousand, Müller M, Braus GH, et al. Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus. Eukaryotic Cell. 2008; 7(8): 1268–1277. pmid:18552284
   • View Article
   • PubMed/NCBI
   • Google Scholar
4. iv. Reignault P, Valette-Collet O, Boccara Grand. The importance of fungal pectinolytic enzymes in plant invasion, host adaptability and symptom type. European Journal of Plant Pathology. 2007; 120(one): 1–11.
   • View Commodity
   • Google Scholar
5. v. Kars I, Krooshof GH, Wagemakers L, Joosten R, Benen JAE, van Kan JAL. Necrotizing activity of v Botrytis cinerea endopolygalacturonases produced in Pichia pastoris. Institute Journal. 2005; 43(ii): 213–225. pmid:15998308
   • View Commodity
   • PubMed/NCBI
   • Google Scholar
6. half-dozen. Kars I, van Kan JAL. Extracellular enzymes and metabolites involved in pathogenesis of Botrytis. Botrytis: Biology, Pathology and Command. 2007; 99–118 p.
7. 7. Jayani RS, Saxena S, Gupta R. Microbial pectinolytic enzymes: a review. Process Biochemistry. 2005; xl (nine): 2931–2944.
   • View Article
   • Google Scholar
8. 8. Lorenzo GD, Ferrari S. Polygalacturonase-inhibiting proteins in defense force against phytopathogenic fungi. Current Opinion in Plant Biology. 2002; 5(four): 295–299. pmid:12179962
   • View Article
   • PubMed/NCBI
   • Google Scholar
9. 9. Shieh MT, Brown RL, Whitehead MP, Cary JW, Cotty PJ, Cleveland TE, et al. Molecular genetic prove for the involvement of a specific polygalacturonase, P2c, in the invasion and spread of Aspergillus flavus in cotton bolls. Practical & Environmental Microbiology. 1997; 63(nine): 3548–3552.
   • View Article
   • Google Scholar
10. x. Accept AT, Mulder West, Visser J, van Kan JAL. The endopolygalacturonase gene Bcpg1 is required for total virulence of Botrytis cinerea. Molecular Plant-Microbe Interactions. 1998; xi(10): 1009–1016. pmid:9768518
    • View Article
    • PubMed/NCBI
    • Google Scholar
11. 11. Valette-Collet O, Cimerman A, Reignault P, Levis C, Boccara Grand. Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants. Molecular Plant-Microbe Interactions. 2003; sixteen(4): 360–367. pmid:12744465
    • View Article
    • PubMed/NCBI
    • Google Scholar
12. 12. Yakoby Due north, Beno-Moualem D, Cracking NT, Dinoor A, Pines O, Prusky D. Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-mucus interaction. Molecular Plant-Microbe Interactions. 2001; fourteen(viii): 988–995. pmid:11497471
    • View Article
    • PubMed/NCBI
    • Google Scholar
13. 13. Wen XS, Yong JJ, Bao ZF, O'Neill NR, Xiao PZ, Xie BY, et al. Functional analysis of Pcipg2 from the straminopilous plant pathogen Phytophthora capsici. Genesis. 2009; 47(eight): 535–544. pmid:19422018
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
14. 14. Isshiki A, Akimitsu Yard, Yamamoto M, Yamamoto H. Endopolygalacturonase is essential for citrus black rot caused by Alternaria citri merely not brown spot acquired past Alternaria alternata. Molecular Establish-Microbe Interactions. 2001; fourteen(6): 749–757. pmid:11386370
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
15. fifteen. Pietro Advertising, Madrid MP, Caracuel Z, Delgado-Jarana J, Roncero MIG. Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt fungus. Molecular Found Pathology. 2003; four(5): 315–325. pmid:20569392
    • View Article
    • PubMed/NCBI
    • Google Scholar
16. sixteen. Di GM FA, R Mig, Di P A. Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum. Molecular Institute-Microbe Interactions. 2000; 13(4): 359–365. pmid:10755298
    • View Article
    • PubMed/NCBI
    • Google Scholar
17. 17. Scottcraig JS, Panaccione DG, Cervone F, Walton JD. Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. Constitute Jail cell. 1990; ii(12): 1191–1200. pmid:2152162
    • View Article
    • PubMed/NCBI
    • Google Scholar
18. eighteen. Oeser B, Heidrich PM, Müller U, Tudzynski P, Tenberge KB. Polygalacturonase is a pathogenicity gene in the Claviceps purpurea /rye interaction. Fungal Genetics & Biology. 2002; 36(3): 176–186.
    • View Commodity
    • Google Scholar
19. xix. de Vries RP, Visser J. Aspergillus enzymes involved in deposition of plant prison cell wall polysaccharides. Microbiology & Molecular Biological science Reviews. 2001; 65(4): 497–522.
    • View Commodity
    • Google Scholar
20. 20. Schuster East, Dunncoleman Due north, Frisvad J, Dijck PV. On the safety of Aspergillus niger–a review. Applied Microbiology & Biotechnology. 2002; 59(4–5): 426–435.
    • View Article
    • Google Scholar
21. 21. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, et al. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnology. 2007; 25(2): 221–231. pmid:17259976
    • View Article
    • PubMed/NCBI
    • Google Scholar
22. 22. Meyer V, Arentshorst Thou, Elghezal A, Drews AC, Kooistra R, Ca VDH, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. Journal of Biotechnology. 2007; 128(4):770–775. pmid:17275117
    • View Article
    • PubMed/NCBI
    • Google Scholar
23. 23. Baltz RH, Davies JE, Demain AL, Baltz RH, Davies JE, Demain AL. Manual of industrial microbiology and biotechnology. ASM Press. 2010.
24. 24. Delmas S, Llanos A, Parrou JL, Kokolski 1000, Pullan ST, Shunburne L, et al. Development of an unmarked factor deletion organization for the filamentous fungi Aspergillus niger and Talaromyces versatilis. Applied & Environmental Microbiology. 2014; fourscore(11): 3484–3487.
    • View Article
    • Google Scholar
25. 25. Miller GL. Use of dinitrosalicylic acrid reagent for determination of reducing sugar. Analytical Chemistry. 1959; 31(three): 426–428.
    • View Article
    • Google Scholar
26. 26. Cotty PJ, Cleveland TE, Dark-brown RL, Mellon JE. Variation in polygalacturonase production among Aspergillus flavus isolates. Applied & Environmental Microbiology. 1990; 56(12): 3885–3887.
    • View Commodity
    • Google Scholar
27. 27. Strauss MLA, Jolly NP, Lambrechts MG, Rensburg PV. Screening for the product of extracellular hydrolytic enzymes by not- Saccharomyces wine yeasts. Journal of Practical Microbiology. 2001; 91(i): 182–190. pmid:11442729
    • View Article
    • PubMed/NCBI
    • Google Scholar
28. 28. Maldonado MC, Strasser dSAM, Callieri DA. Regulatory aspects of the synthesis of polygalacturonase and pectinesterase by Aspergillus niger sp. [pectinase, pectinolytic enzymes, inducible synthesis of enzyme]. Sciences Des Aliments. 1989.
    • View Article
    • Google Scholar
29. 29. Martens-Uzunova E, Zandleven J, Benen J, Awad H, Kools H, Beldman GA, et al. A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation. Biochemical Periodical. 2006; 400(1): 43–52. pmid:16822232
    • View Article
    • PubMed/NCBI
    • Google Scholar
30. 30. Benen JAE, Kester HCM, Visser J. Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. European Journal of Biochemistry. 1999; 259(iii): 577–585. pmid:10092840
    • View Article
    • PubMed/NCBI
    • Google Scholar
31. 31. Pařenicová L, Benen JAE, Kester HCM, Visser J. pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger. Biochemical Journal. 2000; 345(3): 637–644.
    • View Commodity
    • Google Scholar
32. 32. Pařenicová L, Kester HCM, Benen JAE, Visser J. Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties. Febs Letters. 2000; 467(two–3): 333–336. pmid:10675564
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
33. 33. Pařenicová L, Benen JAE, Kester HCM, Visser J. pgaE encodes a quaternary member of the endopolygalacturonase gene family unit from Aspergillus niger. European Journal of Biochemistry. 1998; 251(1–2): 72–80. pmid:9492270
    • View Article
    • PubMed/NCBI
    • Google Scholar
34. 34. Gysler C, Harmsen JAM, Kester HCM, Visser J, Heim J. Isolation and construction of the pectin lyase D-encoding cistron from Aspergillus niger. Gene. 1990; 89(one): 101–108. pmid:2373363
    • View Article
    • PubMed/NCBI
    • Google Scholar
35. 35. Someren KV, Flipphi Chiliad, Graaff LD, Broeck HVD, Kester H, Hinnen A, et al. Characterization of the Aspergillus niger pelB gene: structure and regulation of expression. Molecular and General Genetics. 1992; 234(ane): 113–120. pmid:1495474
    • View Article
    • PubMed/NCBI
    • Google Scholar
36. 36. Harmsen JAM, van Someren MAK, Visser J. Cloning and expression of a 2nd Aspergillus niger pectin lyase factor (pelA): indications of a pectin lyase gene family in A. niger. Current Genetics. 1990; 18(2): 161–166. pmid:2225145
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
37. 37. Solís-Pereira Due south, Favela-Torres East, Viniegra-González G, Gutiérrez-Rojas Thou. Issue of dissimilar carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations. Applied Microbiology & Biotechnology. 1990; 39(i): 36–41.
    • View Commodity
    • Google Scholar
38. 38. Liu N, Ma X, Zhou S, Wang P, Dominicus Y, Li X, et al. Molecular and functional characterization of a polygalacturonase-inhibiting protein from Cynanchum komarovii that confers fungal resistance in Arabidopsis. Plos One. 2015; eleven(1): e0146959.
    • View Commodity
    • Google Scholar
39. 39. Barras F, And VG F, Chatterjee AK. Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annual Review of Phytopathology. 2003; 32: 201–234.
    • View Article
    • Google Scholar
40. 40. Collmer A, Keen NT. The part of pectic enzymes in plant pathogenesis. Annual Review of Phytopathology. 2003; 24: 383–409.
    • View Article
    • Google Scholar
41. 41. Rogers LM, Kim YK, Guo W, González-Candelas 50, Li D, Kolattukudy PE. Requirement for either a host- or pectin-induced pectate lyase for infection of Pisum sativum by Nectria hematococca. Proceedings of the National Academy of Sciences. 2000; 97(17): 9813–9818.
    • View Article
    • Google Scholar
42. 42. Bussink HJD, Buxton FP, Fraaye BA, Graaff LHD, Visser J. The polygalacturonases of Aspergillus niger are encoded past a family of diverged genes. European Journal of Biochemistry. 1992; 208(1): 83–xc. pmid:1511691
    • View Commodity
    • PubMed/NCBI
    • Google Scholar
43. 43. Ramesh MV, Kolattukudy PE. Disruption of the serine proteinase gene (sep) in Aspergillus flavus leads to a compensatory increase in the expression of a metalloproteinase gene (mep20). Journal of Bacteriology. 1996; 178(13): 3899–3907. pmid:8682796
    • View Article
    • PubMed/NCBI
    • Google Scholar

loveexpined.blogspot.com

Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173277

Share this post