Sugarcane: Research Towards Efficient and Sustainable Production. Wilson JR, Hogarth D M , Campbell JA and Garside AL (Eds).
CSIRO Division of Tropical Crops and Pastures, Brisbane. 1996. pp. 135-137 135 ANTIMICROBIAL PROTEINS: NEW OPTIONS FOR DISEASE CONTROL IN SUGARCANE
HARRISON SJ, MARCUS JP, GOULTER KC, BRUMBLEY S, GREEN JL, MACLEAN DJ and MANNERS JM
CRCfor Tropical Plant Pathology, University of Queensland, Q 4072 Australia
A B S T R A C T
An increasing number of proteins that exhibit anti-microbial activity are being isolated from plants. The genes encoding these anti- microbial proteins have potential for the manipulation of heterologous plant systems for enhanced resistance to plant pathogenic microbes. These proteins are categorised into classes or families based on sequence homology or mode of action. In a screen of A ustralian plant accessions significant levels of antimicrobial activity were discovered when soluble protein extracts were bioassayed against the growth of commercially important microbial pathogens in vitro. Two of these extracts were further purified to reveal two low molecular weight cysteine-rich peptides with potent antimicrobial activity toward a panel of phytopathogens including several pathogens of sugarcane. These might be used to increase the resistance of sugarcane to pathogenic organisms.
INTRODUCTION
Current breeding techniques for disease control in plants rely on the existence of natural resistance genes to specific microbial pathogens.
For many of the commercially important microbial pathogens resistance genes are limited or non-existent within the host germplasm. The use of plant transformation technology to introduce a gene or genes encoding resistance determinants will increase the options available to plant breeders for the production of disease-resistant plants. In recent years, many different plant proteins with antimicrobial and/or antifungal activity have been identified and described. These proteins have been categorised into several classes according to either their presumed mode of action and/or their amino acid sequence homologies. These classes include chitinases, b-1,3-glucanases, permatins, ribosome-inactivating proteins, plant defensins, thionins, chitin binding proteins, thaumatin- like, or osmotin-like proteins, PRl-type proteins and the non-specific lipid transfer proteins. There are also other anti-microbial proteins from plants which have not been categorised for example, the anti-microbial proteins of Mirabilis jalapa.
There is already evidence that the expression of genes encoding proteins that have in vitro anti-microbial activity in transgenic plants can result in increased resistance to microbial pathogens. Examples of this engineered resistance include transgenic plants expressing genes encoding: a plant chitinase. either alone (Broglie et al 1991) or in combination with a b-1,3-glucanase (Melchers et al 1993); a plant defensin (Terras et al 1995); an osmotin-like protein (Liu et al 1994) and a ribosome inactivating protein (Logemann et al 1992). The results obtained in vivo for most of these proteins are consistent with the in vitro inhibition experiments performed on the pathogen with purified protein.
The cysteine-rich, basic, low molecular weight antimicrobial proteins are the most potent plant antimicrobial proteins isolated so far. The thionins, whose members have been separated into three classes based on their sequence homology and spectrum of activity are the best characterised of these peptides. The thionins are prevalent in the endosperm of many plant seeds and are believed to have a storage (sulphur) as well as a defensive role. Analogues have been observed in many plant organs including leaf specific isoforms which are stress induced, especially in response to microbial challenge. The a and b thionins are the most toxic of the thionins isolated thus far. They inhibit the growth of gram negative and gram positive bacteria, fungi, insect cells, mammalian cells and some members of these two classes have been shown to inhibit the growth of plant cells (Bohlman et al 1994).
This general toxicity may limit the usefulness of these proteins in transformation work. The third class of thionins are the g-thionins, which also falls under the classification of plant defensins. Like the other members of the thionin family, the g-thionins and plant defensins were initially isolated from seeds. They show inhibitory activity towards fungi and gram positive bacteria but show no inhibition towards the growth of either plant or animal cells (Moreno et al 1994, Osborn et al 1995).
Other cysteine-rich low molecular weight antimicrobial peptides include
the two novel peptides isolated from Mirabilis jalapa and Amaranthus caudatus. These two peptides show no homology to the thionins but show a similar spectrum of inhibitory activity to that observed from the Y-thionins.
Fungal and bacterial pathogens cause considerable losses in sugarcane production each year in Australia. The major diseases include poor root syndrome associated with Pachymetra chaunorhiza and Pythium spp.
and ratoon stunting disease caused by Clavibacter xyli subsp. xyli. Other diseases of either sporadic or lesser importance include pineapple d i s e a s e c a u s e d by Ceratocystis paradoxa, red rot c a u s e d by Colletotrichum falcatum and sugarcane c o m m o n rust caused by Puccinia melanocephala. Sugarcane breeding is a slow and difficult process due to its complex genetics. Durable methods of disease control are desirable to provide protection against the wide range of pathogens which infect sugarcane. Current plant breeding strategies for disease control generally address one disease at a time and are dependent on sources of natural resistance which are limited for many of these diseases. Antimicrobial proteins with broad range activity, that can be expressed directly from suitable constructs in transgenic plants, may be used for the production of disease-resistant sugarcane. This paper describes the screening of seed protein extracts from Australian native plants for antimicrobial activity and the isolation of two novel antimicrobial proteins which inhibit the growth of some sugarcane pathogens.
MATERIALS A N D M E T H O D S Extraction of basic proteins of seeds
Australian native seeds were ground and extracted for 4 hrs at 4°C in 2 volumes of cold extraction buffer (Terras et al 1992). The resulting homogenates were strained through cheese cloth to remove particulate matter and centrifuged at 3,000 x g for 30 min to clarify the solutions.
Solid ammonium sulphate was added to the supernatants to obtain 3 0 % relative saturation and the precipitate allowed to form overnight while gently stirring at 4°C. Following centrifugation at 3,000 x g for 30 min, die supernatants were taken and ammonium sulphate added to achieve 80% relative saturation. The solutions were allowed to precipitate overnight and then centrifuged at 3,000 x g for 30 min in order to collect the precipitated fraction. The 30%-80% fractions were then resuspended in a minimal volume of 20 mM Tris-HCl pH 9 and dialysed overnight at 4°C in the presence of protease inhibitors. After dialysis the protein solutions were passed through anion-exchange columns equilibrated at pH 9 with 20 mM Tris-HCl. The collected flow-through from this column represents the basic (pi >9) protein fraction of the seeds.
Bioassay of protein extracts
All bioassays were carried out in 96-well microtitre plates. Typically, the test organism was suspended in a synthetic growth medium (Terras et al 1992). The test organism consisted of bacterial cells, fungal spores (50,000 spores/mL) or fungal mycelial fragments (produced by blending a hyphal mass from a culture of the fungus to be tested and then filtering through a fine mesh to remove larger hyphal masses). Fifty microlitres
136
of the test organism suspended in medium was placed into each well of the microtitre plate. A further 50 uL of the test anti-microbial solution was added to appropriate wells. To deal with well-to-well variability in the bioassay, 4 replicates of each test solution were performed. Sixteen wells from each 96-well plate were used as controls for comparison with the test solutions. All fungi were grown at 25°C. Clavibacter spp.
was grown at 28°C and E. coli was grown at 37°C. Percent inhibition was measured using an optical density measurement following the c h a n g e in a b s o r b a n c e at 6 0 0 n m . T h e time i n t e r v a l s b e t w e e n measurements were dependent on the organism being assayed. Growth inhibition is defined as 100 times the ratio of the change in absorbance of the average growth in the control wells minus the change in absorbance in the test well over the change in absorbance at 600 nm for the mean of the control wells. The 1C50 value (concentration of which growth was inhibited 50%) was used to compare the activity of protein.
Percent inhibition levels used in the calculation of IC50 values were taken from the second time period (usually 24-48h) in the time course.
RESULTS
Screening for antimicrobial activity
Extracts of soluble basic protein were obtained from the seeds of 200 indigenous Australian plant species. These extracts were screened to assess antimicrobial activity against a panel of important fungal phytopathogens representing the major classes of fungi. T h e panel included; Phytophthora cryptogea, Fusarium oxysporum f.sp. cubense, Sclerolinia sclerotiorum, Sclerotium rolfsii and Colletotrichum gloeosporioides. Screening revealed 20 extracts to show significant inhibitory activity to various members of this panel. The 20 extracts were then screened against a wider range of phytopathogens including several of the major pathogens of sugarcane. Several of the extracts exhibited promising activity against Pythium spp. and Clavibacter spp., and two of these extracts were further characterised. Further purification from the basic extracts of Macadamia integrifolia and Hardenbergia violacea isolated MiAMPl and HvAMPl two novel potent antimicrobial proteins. Mass spectrometric analysis of M i A M P l and Z/vAMPl r e v e a l e d low m o l e c u l a r w e i g h t p r o t e i n s of 8.1kDa and 5.3kDa respectively.
Difficulties were experienced in assaying the antimicrobial proteins against C. xyli subsp. xyli (Cxx) and Pachymetra chaunorhiza. The nutritionally fastidious Cxx w a s subject to contamination in the microplate assay but M i A M P l had significant activity against a closely related bacteria C. xyli subsp. cynodontis. Current work is aimed at developing a viability assay based on light emission using C. xyli subsp.
xyli transformed with luciferase genes. T h e fungus Pachymetra chaunorhiza w o u l d not grow in m i c r o t i t r e p l a t e s . Tests u s i n g antimicrobial peptides applied to wells cut out from agar plates did not indicate any inhibition by the two peptides but more work is needed to develop a better assay system for this fungus.
Antimicrobial activity of ffvAMPl and AfiAMPl The results of bioassays on HvAMVl and AfiAMPl are presented in Table 1. Examples of growth inhibition plots for the proteins against Ceratocystisparadoxa are shown in Fig. 1. Both AfiAMPl a n d / / v A M P l are potent inhibitors of the in vitro growth of many of the major pathogens of sugarcane as well as other crops. The anti-microbial activity is greatly reduced in the presence of the divalent cation (Ca2*).
Similar reductions in potency in the presence of 1 mM Ca2" have been seen with other anti-microbial proteins (Terras et al 1992). Significant morphological changes were observed when several of the ascomycetes were treated with H v A M P l and AfiAMPl. Increased branching and swelling of the hyphae was typically seen in the presence of the proteins.
DISCUSSION
Through the screening of 200 basic protein extracts we have isolated 20 extracts with antimicrobial activity against many commercially important phytopathogens. Two of these extracts have been further purified to reveal two novel antimicrobial proteins designated / / v A M P l and AfiAMPl. Both peptides have been demonstrated to be potent inhibitors of the growth of many microbial phytopathogens in vitro.
Table 1 The IC50 value (mg/mL) of HvAMPl and MiAMPl against various fungal and bacterial pathogens of sugarcane, as well as several other commercially important fungal pathogens.
[The results for the fungal pathogens presented in the first column under
" A " , were obtained using the synthetic low ionic strength growth medium (Terras et al. 1992). The antimicrobial protein was also tested in the presence of 1 mM Ca2* and 50mM KCl in the test medium. The results for these experiments are presented in the column headed " B " . The bacterial pathogen was growth in S8 media ("A") and S8 media supplemented with 1 mM Ca2* and 50mM KCl ("B"). Clavibacter xyli subsp. cynodontis was used in preference to C. xyli subsp. xyli because of its faster growth rate. ND means the experiment was not done.]
Pythium graminicola 50 5 >100 >100 Ceratocystis paradoxa 10 20 >100 75 Colletotrichum falcatum >100 >100 >100 >100 Clavibacter xyli
subsp. cynodontis ND 10 ND 10 Alternaria helianthi 5-10 2-5 75 >100 Sclerotinia sclerotiorum 20 5 100 >100
0 20 40 60 80 100 Concentration of HvAMPl (ug/mL)
0 20 40 60 80 100 Concentration of MiAMPl (ug/mL)
Fig. 1 Growth inhibition % of Ceratocystis paradoxa treated with proteins (a) HvAMPl and (b) MiAMPl over a 12 to 24 h period in medium A and B.
[Medium A is a synthetic low ionic strength growth medium (Terras et al 1992), medium B is medium A supplemented with 1 mM Ca2* and 50mM KCl.]
137 These results identify MiAMP I and HvAMPl as possible candidates
for the production of transgenic plants expressing these two proteins.
Amino acid sequences have been determined for both proteins and attempts are currently underway to clone cDNAs corresponding to the M i A M P l and H v A M P l proteins. Once cloned, plant expression constructs can be made to examine the activity of these proteins in vivo. Initial experiments will involve their expression in model plant systems such as tobacco or Arabidopsis. Their ability to enhance resistance to phytopathogens will be assessed after transforming commercially important plant species. The ultimate aim of the work is to express genes corresponding to these proteins in sugarcane. Plant expression constructs will be produced under the control of monocot organ-specific promoters dependent on the target disease eg. vascular- expressing promoter for Clavibacter xyli subsp.xyli, leaf-expressing promoter for Puccinia melanocephala or root-expressing promoter for Pachymetra chaunorhiza.
A number of basic protein extracts which exhibited potent antimicrobial activity are yet to be investigated. Attempts are currently underway to purify the active components of these protein extracts with the aim of broadening the arsenal of antimicrobial proteins. If proteins could be isolated with either a broader range or higher level of activity they could be important in producing cassettes containing numerous peptides, that could be concurrently expressed to provide a broader range of resistance.
At present most antimicrobial proteins under development have been identified by overseas multinational corporations. The use of these genes/proteins in Australian agriculture will be under terms dictated by these off-shore companies. The identification of new proteins in an Australian institution will permit less encumbered applications.
A C K N O W L E D G E M E N T S
We are grateful to the SRDC for a Postgraduate Research Scholarship for Stuart Harrison and for a one year pilot research grant. We thank Dr. C. Grof for generous access to protein purification equipment supplied by the SRDC.
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Melchers LS, Sela BMB, Vloemans AS, Woloshuk CP, Roekel JSCv, Pen J, Elzen PJMvd, Cornelissen BJC (1993) Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and beta-1,3- glucanase in transgenic plants. Plant Molecular Biology 21 583- 593.
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Osbom RW, De SG, Thevissen K, Goderis I, Torrekens S. Van LF, Attenborough S, Rees SB, Broekaert WF (1995) Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Letters 368 257-62.
Terras FR, Schoofs HM, De BM, Van LF, Rees SB, Vanderleyden J, Cammue BP, Broekaert WF (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.
Journal Biological Chemistry 267 15301-9.
Terras FR, Torrekens S, Osborn RW, Vanderleyden J, Eggermont K, Kovaleva V, Raikhel NV, Kester A, Rees S B , Torrekens S, Van Leuven, Cammue BPA, Broekaert WF (1995) Small cysteine-rich antifungal proteins from radish: their role in host defense. The Plant Cell 7 573-588.
Sugarcane: Research Towards Efficient and Sustainable Production. Wilson JR, Hogarth D M , Campbell JA and Garside AL (Eds).
138 CSIRO Division of Tropical Crops and Pastures, Brisbane. 1996. p p . 138-140
GENETICALLY ENGINEERING RESISTANCE TO SUGARCANE MOSAIC AND FIJI DISEASE VIRUSES IN SUGARCANE
SMITH GR1, JOYCE PA1, HANDLEY JA2, SITHISARN P2, MAUGERI MM-, BERNARD MJ1, BERDING N3, DALE JL2 and HARDING RM2
1.David North Plant Research Centre, BSES, PO Box 86, lndooroopilly Q 4068, Australia 2.Centre for Molecular Biotechnology, QUT, George St, Brisbane Q 4001, Australia 3.Meringa Sugar Experiment Station, BSES, PO Box 122, Cordonvale Q 4865, Australia
ABSTRACT
Two important pathogens of sugarcane are sugarcane mosaic and Fiji disease viruses. These viruses can cause significant yield losses in susceptible crops, while their potential presence can restrict the extent of cultivation of sugarcane clones or affect the choice of parents in plant improvement programs. Backcrossing to introduce resistance genes is not practical in sugarcane due to the complex polyploidy and heterozygosity of the genome. Pathogen-derived resistance (PDR) is being exploited successfully in other crops to produce transgenic virus resistant clones. The coat protein coding region of sugarcane mosaic virus (SCMV) has been cloned and developed into a gene suitable for sugarcane transformation. Transgenic sugarcane plants containing this coat protein are being evaluated. Further PDR genes for resistance to SCMV are being developed, which will allow full exploitation of potential transgenes and the possibility of pyramiding genes into transgenic plants: similar work is underway to develop PDR genes from the Fiji disease virus genome. Novel genes for resistance to these viruses will produce significant gains for the sugar industry.
INTRODUCTION
Commercial cultivation of sugarcane (Saccharum L. spp. hybrids) is affected by a wide range of viral, fungal and bacterial pathogens. The main viral pathogens of sugarcane present in Australia include sugarcane mosaic potyvirus (SCMV). Fiji disease reovirus (FDV) and sugarcane bacilliform badnavirus (SCBV). The recently reported yellow leaf syndrome is probably caused by a luteovirus, while chlorotic streak is probably of viral or viroid aetiology. In general, there is resistance to these pathogens within the 'Saccharum c o m p l e x ' gene pool, but introducing resistance into agronomically elite clones has proved difficult because of the complex genetics of sugarcane. Backcrossing to introduce specific genes is virtually impossible due to the uncertain chromosome composition of commercial clones and the aneuploid nature of the progeny.
Resistance against viruses from a wide range of families, including the potyviridae (Clough & Hamm 1995) and the luteoviridae (Wilson 1993), is being successfully engineered into many crop plants. Work is currently underway to engineer resistance to reoviruses such as rice dwarf and rice ragged stunt (Matsummura & Tabayashi 1995). These resistance genes originate from the pathogen itself, and are usually referred to as pathogen-derived resistance (PDR) genes (Sanford &
Johnstone 1985). Following the first practical demonstration of the concept by Powell Abel et al (1986), transgenic plants have been produced carrying full length, truncated, untranslatable or mutated versions of coat proteins, replicases, movement proteins, and other sequences of viral origin. A genetic construct based on the full length coat protein of one Australian isolate of SCMV strain A (SCMV-A) was developed and expression of this gene demonstrated in sugarcane protoplasts (Smith et al 1992). However, there are questions about the basic underlying principle of coat protein mediated resistance (Wilson 1993; Smith et al 1995b). There is evidence that some transgene- mediated resistance acts at the RNA and not the protein level, and that r e s i s t a n c e r e s u l t s from the a c t i v a t i o n of natural p l a n t defence mechanisms. Genetic variability in the pathogen also can significantly influence the effectiveness of transgenic resistance. In some instances, very small differences in the nucleic acid sequence between the transgene and the infecting virus can result in no resistance to the pathogen (Wilson 1993). However, there are also examples where transgenic r e s i s t a n c e holds even though there are c o n s i d e r a b l e differences between the nucleic acid sequences (Wilson 1993).
Meaningful exploitation of PDR genes for sugarcane genetic engineering requires knowledge and clones of the pathogen nucleic acid so that informed decisions can be made about the type of resistance transgene that could be developed. Here, we present progress on the development
of SCMV-resistant transgenic sugarcane, the selection of further genes for S C M V r e s i s t a n c e , analysis of p a t h o g e n variability, and the development of PDR genes for resistance to FDV.
MATERIALS A N D M E T H O D S SCMV transgenics
The coat protein (CP) coding region of SCMV-A was cloned by Frenkel et al. (1991) and subsequently modified into a gene and constructed into an expression vector driven by the Emu promoter (Smith et al 1992).
This gene subsequently was placed under control of the maize ubiquitin promoter (Ubi) in both sense and anti-sense directions. Induction of embryogenic callus, microprojectile transformation, and selection/
regeneration of plantlets were essentially as described by Bower & Birch (1992). Analysis of regenerated plants by PCR, western and Southern blots were essentially as described by Smith & Gambley (1993).
Analysis of S C M V field isolates
Leaves infected with SCMV were collected from the BSES Pathology Farm (Brisbane) and near Bundaberg. Childers (Isis Mill area) and Nambour in south east Queensland. Total nucleic acids were extracted, and viral specific RNA was amplified essentially by the reverse transcription-polymerase chain reaction (RT-PCR) protocol described by Smith & Van de Velde (1994). The amplified products were cloned using the pGEM-T Vector System (Promega) and three independent clones from each sample were cycle-sequenced to eliminate errors due to PCR amplification. The RT-PCR and cycle-sequencing conditions and generation of sequence data were essentially as described by Smith e t a l (1995a).
Generation and analysis of FDV clones
Galls were cut from FDV-infected leaves of sugarcane maintained at the BSES Pathology Farm, Eight Mile Plains. Double-stranded RNA (dsRNA) was extracted and then primed for reverse transcription by boiling the dsRNA and hexanucleotide primers together for 8-10 min followed by quenching in dry ice/ethanol or liquid nitrogen. Second strand synthesis was by a combination of D N A polymerase I and RNaseH, and after the ends were polished, the cDNA was cloned into the Smal site of pUC18 or pGEM-3Zf+. The ligation reaction was transformed into competent E. coli JM109 cells and clones containing recombinant plasmids selected by blue/white differentiation on IPTG/
X-gal supplemented media. Plasmids were prepared from selected clones, labelled with 32P by random priming and used as hybridisation probes against northern blots of FDV dsRNA to identify the segment from which the cloned cDNA originated. Clones unique to individual FDV segments were cycle-sequenced as described above, and the sequences aligned and compared using programs maintained on the