Rational Environmental Management of Agrochemicals - American

Chapter 19

Antifungal Antibodies in Plant Pathology 1

Claudia Sheedy and J. Christopher Hall

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Downloaded by FUDAN UNIV on February 25, 2017 | http://pubs.acs.org Publication Date: September 21, 2007 | doi: 10.1021/bk-2007-0966.ch019

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Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, 5403 First Avenue South, Lethbridge, Alberta T1J 4B1, Canada Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

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For several decades, polyclonal and monoclonal antibodies, and more recently recombinant antibodies and fragments thereof have been used for pesticide residue analysis and biomedical applications. Antibodies are being used now to treat cancer, and have been produced in plants for protection against viral and fungal pathogens, as well as for pesticide resistance. This article comprises a review of anti-fungal monoclonal, polyclonal and recombinant antibodies used in plant pathology over the last two decades. The antigens used for animal immunizations, the specificity of the antibodies thereby obtained, and a few examples of practical applications of these antibodies such as immunodiagnostics, immunofluorescence, fungal growth inhibition, aerobiology and glycobiology studies are reviewed.

Introduction In plant pathology, immunological methods have been mainly used to detect or identify plant viruses and fungi ( 7 ) . One of the greatest hurdles to further use of anti-fungal antibodies in plant pathology has been their lack of specificity ( 7 , 2 ) . Yet, phytopathogenic fungi are responsible for extensive losses in food, food storage and food distribution ( 3 ) . Moreover, the presence of fungi can seriously affect the nutritional value, colour, flavour and texture of grains and plant 306

© 2 0 0 7 American Chemical Society

Kennedy et al.; Rational Environmental Management of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


307 products for example, and the production of mycotoxins may pose a health risk to human populations (2). Antibodies to a great variety of fungi and/or fungal antigens have been raised, most often, to develop enzyme-linked immunosorbent assays (14,5,6,7,8,9,10,11,121 especially for the detection of molds (13,14,15,16,17). However, the elucidation of taxonomic relationships among fungal species (5), the identification of common structures in different fungal species (18,19), disease prediction modelling and aerobiology (20,21) as well as the understanding of fungal biology, glycobiology, morphology and host/pathogen interactions at the molecular level ( 2 2 ) are other applications of antibodies in plant pathology. A comprehensive list of polyclonal and monoclonal antibodies against plant pathogenic fungi are given in Tables I and II, along with the antigens used for immunizations, the antibody specificity and application. Each type of antibody (polyclonal, monoclonal, recombinant) has its own advantages and disadvantages: Polyclonal antibodies are easy to produce, but possess multiple specificities and are not reproducible. Monoclonal antibodies on the other hand, are issued from a single clone with one unique specificity. However, they require expertise and are costly to produce. Recombinant antibodies finally, are isolated along with their respective genes and can therefore be easily manipulated. However, they tend to be, especially in the case of antibodyfragments,less stable than their parental, full-size counterparts.

Fungal Antigens for Animal Immunizations Fungal immunogens used to raise antibodies have been described in several excellent review articles (1,4,5) as well as fungal antigen preparation (12,23,24,25) and their binding to microtiter plate wells for immunoassay applications (75). A variety of fungal materials have been used for the production of fungal-specific antibodies, among which the most commonly used are mycelium and mycelium-derived immunogens. Others are zoospores, spores, conidia, ascospores, cysts, as well as glycoproteins, exoantigens and polysaccharides.

Anti-fungal Antibodies Specificity and Affinity Antibody specificity has been the greatest hurdle in the past to further use of antibodies in plant pathology. This has to be circumvented to make ELISA amenable to routine analysis of fungal infection of plant material, food and other environmental matrices. Overall, antibodies raised against the fungal mycelium have lower specificity compared to antibodies raised against zoospores and



308 cysts. Although such cross-specificity may be of some interest, in general higher specificity is required for the accurate detection and quantification of fungal pathogens or antigens. Carbohydrate antigens seem to be fairly good immunogens, leading to specific antibodies, as high as race-specific in some cases (69). However, several fungi genera may display similar carbohydrate structures on their surfaces. This is the case with Aspergillus and Penicillium conidia and mycelia, which both display galactomannans at their cell surface (70). When a monoclonal antibody specific for galactomannan was tested for cross-reactivity to other fungi however, it showed very little cross-reactivity except for these two other genera (70). Several antibodies raised against fungi appear to bind to carbohydrate epitopes at the surface of these fungi (70,71,72) or to carbohydrate moieties specific to a layer of the fungi surface (70).

Use of Anti-fungal Antibodies in Plant Pathology Antibodies possess several advantages over traditional methods and conventional assays to identify, quantify and neutralize fungal pathogens. One of these advantages is that antibodies can detect fungal pathogens even before the appearance of disease symptoms (57). Antibodies can be used for a wide range of applications in plant pathology: not only can they be useful in fungal immunodiagnostics, immunofluorescence and immunoblotting, but also in neutralizing fungal growth, as a tool to predict fungal infection as well as a tool to study fungal morphology, biology and reproduction. More recently, antibodies have even been expressed in planta for resistance against fungal diseases.

Fungal Immunodiagnostics Most antibodies raised against fungal antigens have been used to develop immunodiagnotic tools such as ELISA or other antibody-based assays. Routine methods for fungal detection are based on plate cultures, microscopy and chemical techniques, but these methods have low specificity and are timeconsuming compared to immunotechniques (37). Immunodiagnostics of fungal pathogens are important, allowing for the detection of potentially harmful toxins in food as well as better disease management strategies. Immunoassays to many different fungal pathogens have been developed: soil-borne and root-infecting fungi such as Phytophthora fragariae (60) and Pythium ultimatum (73), foliar fungi such as Phytophthora infestans (61), seedborne fungi such as Pyrenophora spp. (74), spores of airborne fungi such


309 as Pyricularia grisea (75) as well as postharvest spoilage-causing fungi such as Botrytis cinerea (33,61), to give only a few examples (reviewed in 1).


Immunofluorescence Several anti-fungal polyclonal antibodies (50,44) as well as monoclonal antibodies (72) have been used in immunofluorescence studies. For example, polyclonal antibodies produced against the soluble and cell-wall antigens of Phaeolus schweinitzii (50) and Mycena galopus (44). P. schweintzii is responsible for root and butt-rot in a number of conifer species. Polyclonal antibodies raised against the mycelium of Fusarium oxysporum, a fungus responsible for root rot in soybean, have been used to detect by immunoassay and locate by immunostaining common antigens shared by the host and pathogen (42). The antigens could not be identified, but were detected around the xylem elements, endodermis and epidermal cells of soybean's root sections, and also present on the mycelium, microconidia, macroconidia and chlamydospores of the fungus (42). A possible role of these common antigens as a basic factor in the compatibility between host and pathogen was investigated, since differential fluorescence in the resistant and susceptible cultivars examined was observed, as previous studies had suggested (42).

Fungal Growth Inhibition and Plant Protection Three monoclonal antibodies have been produced against the endophytic fungus Neotyphodium ceonophialum (3). These monoclonal antibodies were incorporated into fungal culture medium to determine their effect on fungal growth (3). These Neotyphodium-specific antibodies inhibited fungal growth, and microscopy revealed that upon binding of the monoclonal antibodies to the fungus, the latter changed its morphology with clumps and few hyphal growth (3). The monoclonal antibodies seem to bind specifically to a cell-wall bound component of the mycelium, but its function remains unknown (3). Antibodies expressed in plants could protect the latter against various pathogens. Several antibodies have been expressed in plants already, a few of which targeted for plant protection against pathogens such as viruses (76,77,78) and mollicutes (79). In this work, three phage-display libraries were created with the mRNA from chickens immunized with three different Fusarium graminearum antigens: cell-wall bound proteins, mycelium surface proteins and germinated spores (80). The libraries were pooled and screened for the isolation of cell-wall specific antibodies over three rounds of panning (80). The recombinant antibody isolated was fused to one of three antifungal peptides, and


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Table I: Polyclonal antibodies against plant pathogenic fungi Fungus Aspergillus spp. Alternaria spp. A. alternata A. ochraceus A. versicolor Botrytis spp. Botrytis cinerea Botrytis fabae Cladosporium cladosporioides C. herbarum Fusarium spp. Fusarium graminearum & F. moniliforme F. moniliforme F. oxysporum F. oxysporum F. oxysporum F. oxysporum F. oxysporum F. poae F. sporotrichoides Gaeumannomyces candidum G. graminis Macrophomina circinelloides M. phaseolina Monascus spp. Mycena galopus Mycosphaerella brassicicola Ophiostoma ulmi P. aurantiogriseum P. aurantiogriseum P. chrysogenum

Antigen M M M ExAg EPS M M M EPS M EPS M ExAg, M

Specificity + + + +++

M EPS EPS M Conidia M ExAg, M ExAg EPS

+ ++ ++ + +++ ++ +++ ++ ++

+ + + + +++ ++ + ++

Usage Taxonomy IF detection ELISA ELISA ELISA Detection Detection Immunocytochemistry Glycobiology ELISA ELISA ELISA, IB

ELISA ELISA Glycobiology ELISA ELISA ELISA, IF ELISA, IB ELISA, IB ELISA

M EPS

+ +

Detection ELISA

M M M Ascospore

+ + + +

ELISA Detection IF Aerobiology

M ExAg M EPS

Reference 18 26 27,28 29,30 31 32 33 34 35 31 36 37

40 25 25,38 39 41 42 37 37 31 43 26 32 32 44 20,21

+++ +++

ELISA ELISA

45 46

+

ELISA

14

++

ELISA

31


311


Table I: Continued. Antigen EPS M M

M M M M zoospores

+ ++ + +

P. fragariae Phytophthora infestans Plasmopara halstedii Postia placenta

M M

+ +

M

+

ELISA Taxonomy ELISA ELISA Dip-stick assay and structural biology studies ELISA Fungal biomass estimation ELISA

PM

+

Detection assay

62

+++ + +++

ELISA ELISA ELISA

63 64 65

M ++

+++ ELISA

27,28 66

+

ELISA

67

++

ELISA

68

M

xylanase Pyricularia oryzae M Pythium violae M Rhizoctonia Toxin solani R. stolonifer M Sclerotinia ExAgs sclerotiorum spores Spongospora subterranea Tilletia indica M

Specificity Usage ++ Detection assay + Taxonomy + Detection

Reference 47 48 49

Fungus P. digitatum P. verrucosum Peronospora viciae Phaeolus schweinitzii Phomopsis spp. Phytophthora spp. Phytophthora spp. Phytophthora spp. Phytophthora cinnamomi

+++

50

IF

23,51,52 53 54 55 56,57,58,59

EPS : Extracellular polysaccharide

+++: Species-specific

ExAg: exoantigens

++: Genus-specific

M : Mycelium/mycelium-derived antigen(s)

+: Less than genus-specific

60 61 61


312 expressed in planta (80). Expression of the antibody fused to any of the three antifungal peptides resulted in a high level of protection against Fusarium oxysporumdue to interference with fungal growth in planta (80). Such results show that antibodies may prove an alternative for the production of transgenic crop plants resistant to fungal pathogens (80).


Table II: Monoclonal antibodies against plant pathogenic fungi Usage Specificity ? ELISA ? ELISA ? Isolation

Fungus Botrytis cinerea Botrytis cinerea Collecotrichum lindemuthianum Fusarium oxysporum Fusarium spp. Gliocladium roseum

Antigen ExAg conidia M M M M

+ +++ +++

Humicola lanuginosa Microdochium nivale Neotyphodium coenophialum Ophiostoma ulmi Penicilium aurantiogriseum P. bilaii P. brevicompactum P. frequentans P. islandicum Phytophtora cinnamomi

M M M

P. megasperma Plasmopara halstedii Postia placenta Pyrenophora graminea Pythium sulcatum P. ultimatum Rhizoctonia solani Trichoderma

Reference 81 24,82 83,84 85 86 71

+ +++ +++

Detection Detection? colonization studies ELISA ? Growth control

3,87 86 3

M M

+++ +

ELISA ELISA

2,45 88

Galactomannan Spores EPS ExAg Zoospores, cysts

+ + +++ + +++

Glycoprotein M B-l,4-xylanase ?

M ? M Glycoprotein

+++ + ? + +++ ? ++

9

Aerobiology ? ELISA Surface component ID Detection ELISA Capture assay ELISA ELISA ELISA IF, detection IF, detection

EPS : Extracellular polysaccharide

+++: Species-specific

ExAg: exoantigens

++: Genus-specific

M : Mycelium/mycelium-derived antigen(s)

+: Less than genus-specific


89 90 70 6,91,92 72 93 94 62 74 95 73 96,97 98-102

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Aerobiology Detection of airborne fungal spores represents a major challenge in plant pathology and epidemiology (90,103). Detecting the source and importance of spore inoculum in the field could be of great assistance in order to achieve efficient use of fungicides, both in terms of timing and quantities (20). Kennedy et al. (20) produced polyclonal antibodies against the ascospores of Mycosphaerella brassisicola, a fungus responsible for ringspot, a major foliar disease of vegetable brassicas. The antibodies were used in conjunction with an existing spore trapping system to detect ascospores by immunofluorescence (20). Although relatively specific, the antibodies cross-reacted with the condia of several fungal species (Pyrenopeziza brassicae, Mycosphaerella pinodes and Cladosporium herbarum) and wall components of ascospores of G. graminis, M. pinodes and P. brassicae (21). Another method to monitor airborne inoculum was developed and investigated by Kennedy et al. (21). The system consisted in a microtiter immunospore trapping device where a suction system actively sucked air particles and impacted them onto microtiter plate wells. The system was tested with two fungal pathogens commonly found in brassicafields,M. brassisicola and Botrytis cinerea, and polyclonal antibodies developed against ascospores of M. brassisicola and conidia of Botrytis cinerea (21). Airborne inoculum could thereby be sampled in the field and targeted organisms rapidly and accurately quantified without any microscopic examination being required (21).

Fungal Pathogen Biology, Glycobiology and Taxonomic Studies Immunological techniques have been used to study several aspects of plant pathology such as biology, disease physiology (34,65) and fungal glycobiology (19,22). Knowledge gainedfromthese studies can improve our understanding of some processes involved in fungal growth morphology (104), reproduction (58) as well as host/pathogen interactions (68). Polyclonal antibodies developed against mycelial antigens of Tilletia indica (causing agent of karnal bunt in wheat) were used to investigate differential expression of proteins or epitopes at the surface of the pathogen at different stages of its development (68). It was demonstrated that several epitopes were modulated during growth and sporulation of T. indica (68). The identification of epitopes specific to species and genera can be very useful in fungal taxonomy as well (42,56,92).

Recombinant Antibodies Recombinant antibody technology has been developed in the 1990s, and opens new possibilities of great interest for plant pathologists. Animal



314 immunization is no longer necessarily required, antibodies against virtually any fungal antigen can be obtained in a few weeks from naive libraries, or a few months from immune libraries, and these antibodyfragmentscan be expressed in bacteria such as Escherichia coli to obtain an endless supply of consistent material. For a full review of recombinant antibody technology see 105,106. With recombinant antibody technology, antibodies andfragmentsthereof are displayed at the surface of filamentous bacteriophage (phage-display antibody libraries), yeast (yeast-display) or ribosomes (ribosome-display). The display technology allows us to select, or isolate, an antibody of interestfromthe library by a process generally referred to as "panning", where the antibody library is exposed to the antigen, and antibodies displayed which bind to this antigen can be isolated from the rest of the library and then expressed in bacteria, plants or yeast. The advantage of these display technologies is that the phenotype (the antibody protein) is linked to the genotype (antibody DNA), so that both are selected simultaneously. Moreover, stringent antibody selection strategies can isolate clones highly specific for a single fungal species, based on the panning strategy used. Panning only requires a few weeks (once the library has been constructed), and can result in antibody clones with greater affinity and specificity compared to those of polyclonal and monoclonal antibodies.

Conclusion Polyclonal and monoclonal antibodies, and more recently recombinant antibodies have all been used in plant pathology. These antibodies have shown various degrees of specificity, depending mainly on the fungal immunogen used for animal immunizations: mycelium and mycelium-derived antigens, zoospores, ascospores, conidia, cysts, and fungal surface components such as glycoproteins, exoantigens and polysaccharides. The antibodies have been successfully used to develop immunodiagnostics and tools for fungal immunofluorescence, fungal identification, growth inhibition, disease prediction modelling and to study the plant pathogen's biology, reproduction and glycobiology. However, with the advent of recombinant antibody technology, where highly specific antibodies against any fungal plant pathogen or its components can be rapidly isolated, novel and exciting applications in plant protection and disease prediction can be foreseen.

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