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First published online August 23, 2002; 10.1105/tpc.003426 American Society of Plant Biologists Two Novel Fungal Virulence Genes Specifically Expressed in Appressoria of the Rice Blast Fungus
a Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 1 To whom correspondence should be addressed. E-mail xu{at}btny.purdue.edu; fax 765-494-0363
The PMK1 mitogen-activated protein kinase gene regulates appressorium formation and infectious hyphae growth in the rice blast fungus. To further characterize this mitogen-activated protein kinase pathway, we constructed a subtraction library enriched for genes regulated by PMK1. Two genes identified in this library, GAS1 and GAS2, encode small proteins that are homologous with gEgh16 of the powdery mildew fungus. Both were expressed specifically during appressorium formation in the wild-type strains, but neither was expressed in the pmk1 mutant. Mutants deleted in GAS1 and GAS2 had no defect in vegetative growth, conidiation, or appressoria formation, but they were reduced in appressorial penetration and lesion development. Interestingly, deletion of both GAS1 and GAS2 did not have an additive effect on appressorial penetration and lesion formation. The GAS1–green fluorescent protein and GAS2–green fluorescent protein fusion proteins were expressed only in appressoria and localized in the cytoplasm. These two genes may belong to a class of proteins specific for filamentous fungi and function as novel virulence factors in fungal pathogens.
The ascomycete Magnaporthe grisea is pathogenic to economically important crops such as barley, wheat, rice, and millet. Rice blast, which is caused by this heterothallic haploid fungus, is one of the most severe fungal diseases of rice throughout the world (Valent, 1990  ). Genetic studies of this pathogen during the past decade have made the Magnaporthe–rice pathosystem an excellent model for investigating fungus–plant interactions.
Magnaporthe infects rice plants with specialized infection structures called appressoria. Enormous turgor pressure generated in appressoria by the accumulation of high concentrations of glycerol is the force used to penetrate the underlying plant surface (de Jong et al., 1997
Appressorium formation and penetration processes have been studied extensively in Magnaporthe (Dean, 1997
Like pmk1 mutants in Magnaporthe, gene replacement mutants of PTK1 in P. teres and CMK1 in C. lagenarium are nonpathogenic and fail to colonize healthy or wounded host tissues (Takano et al., 2000 Although PMK1 homologs have been identified in several fungi, it is not clear how PMK1 is activated and what kinds of genes are regulated by this MAP kinase pathway. In this study, we constructed a subtraction library enriched for genes regulated by PMK1 during appressorium formation. Two genes identified in this library, GAS1 and GAS2, also were isolated by screening of an appressorium cDNA library by differential hybridization. Both of them were expressed specifically during appressorium formation and localized in the cytoplasm of appressoria. Gene deletion mutants of GAS1 and GAS2 had no defect in growth, conidiation, germination, or appressorium formation but were reduced in penetration and virulence on rice and barley seedlings.
Identification of the gEgh16 Homologs GAS1 and GAS2 To identify genes regulated by PMK1 during appressorium formation, we constructed a subtraction library using Guy11 cDNA as the tester and nn78 cDNA as the driver. Sequence analysis with the first 96 clones of this library revealed that two of them, MBC4 and MBE5, are homologous with the Erysiphe graminis EST clone gEgh16, a protein expressed during the early infection stage (Justesen et al., 1996 ). Interestingly, MBC4 and MBE5 were found to correspond to MAS1 and MAS3, respectively. MAS1 and MAS3 are the most redundant and the third most redundant MAS (Magnaporthe appressoria specific) sequences identified in an independent differential hybridization analysis of an appressorium cDNA library constructed from strain 70-15. Among 621 cDNA clones that exhibited upregulated expression during appressorium formation, 77 and 53 clones are represented by MAS1 and MAS3 sequences, respectively. The 0.35- and 0.4-kb inserts amplified from clones MBE5 and MBC4, respectively, were used as probes to screen a Guy11 cosmid genomic library. Two corresponding open reading frames were identified after sequencing of cosmid clones that hybridized to these two probes; they were named GAS1 and GAS2 (gEgh16 homologs expressed in appressorium stage). The GAS1 gene contains one 63-bp intron and encodes a 251–amino acid protein. The GAS2 gene has no intron and encodes a 290–amino acid protein. The amino acid sequences of GAS1 and GAS2 share 42% identity, and both are homologous with gEgh16 and its homolog Egh16H1. The highest homology is between GAS2 and Egh16H1 (67% identity). The Gas1 and Gas2 proteins are rich in Gly (12.7 and 12.6%, respectively) and Ala (12.0 and 15.8%, respectively) residues. Many Gly and Ala residues are conserved among GAS1, GAS2, and their homologs (Figure 1).
Putative homologs of GAS1 and GAS2 also were found in the Neurospora crassa genome sequenced at the Whitehead Research Institute (www-genome.wi.mit.edu/annotation/fungi/neurospora/), in the Aspergillus fumigatus genome (tigrblast.tigr.org/ufmg), and in the ESTs of the entomopathogenic fungus Metarhizium anisopliae from the cDNA library of infected insect cuticles. However, none of these Gas1/Gas2 homologs has been characterized functionally, and no other close homologs of Gas1/Gas2 with an identified function were found in BLAST searches. Interestingly, there is no homolog of GAS1 or GAS2 in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, or other sequenced eukaryote genomes, indicating that these two genes are specific for filamentous fungi.
Both GAS1 and GAS2 Are Expressed Specifically at the Appressorium Formation Stage
To further confirm that GAS1 and GAS2 are expressed specifically during appressorium formation, RNAs from ungerminated conidia, conidia germinated in liquid 5 x YEG for 4 h, and rice leaves infected with Guy11 for 60 h and 5 days were isolated. No detectable hybridization signal was observed in RNA gel blots of these RNAs hybridized with GAS1 and GAS2 (data not shown). Most likely, GAS1 and GAS2 are expressed only during appressorium formation in Magnaporthe. Approximately 1.2- and 1.4-kb upstream sequences of GAS1 and GAS2 were sequenced and analyzed with several programs, including TRES (www.bioportal.bic.nus.edu.sg/tres), Expasy (www.expasy.org), and SoftBerry (www.softberry.com). Several pyrimidine-rich repeat sequences (CT box) were located downstream from the putative TATA boxes at -257 in GAS1 and -230 in GAS2. The promoter region of GAS1 also has a putative CAAT box (-276) upstream from the start codon. Even though both GAS1 and GAS2 are expressed specifically in appressoria, no other known sequence element was found to be common in their promoter areas.
gas1 Mutants Are Defective in Penetration
The gas1 deletion mutants BC7 and BC46 had no obvious defects in vegetative growth, conidiation, or sexual reproduction, and they formed typical grayish wild-type colonies. Conidia produced by these mutants were normal in germination and appressorium formation (Table 1). Appressoria formed by gas1 mutants were melanized and regular in shape (Figure 4). However, the percentage of appressoria that penetrated onion epidermal cells was reduced in the gas1 mutants. Although >70% of Guy11 and BC32 appressoria penetrated and developed infectious hyphae after 48 h of incubation, only  25% of appressoria formed by gas1 mutants penetrated under the same conditions (Table 1, Figure 4). Thus, the majority of appressoria formed by gas1 mutants were defective in appressorial penetration.
gas1 Mutants Are Reduced in Virulence Rice leaves of CO39 seedlings sprayed with conidia of gas1 mutants developed blast lesions 7 days after inoculation (Figure 5A). However, the number of lesions caused by the gas1 mutant BC46 was much lower than that caused by Guy11 under the same conditions (Table 1). On average, Guy11 caused 45 lesions per 5-cm leaf tip, whereas leaves inoculated with BC46 conidia had 13 lesions per 5-cm leaf tip (Table 1), an 70% reduction compared with Guy11. On barley leaves inoculated with BC46, the number of lesions formed also was reduced (Figure 5B). In addition, lesions formed by BC46 on rice or barley leaves usually were smaller and less spreading than the lesions formed by Guy11 (Figure 5A). A similar reduction in lesion formation and lesion size also was observed in the gas1 deletion mutant BC7 (data not shown).
For cosegregation assays, we isolated and characterized 24 progeny from a cross between BC46 and 2539. Although all 12 hygromycin-resistant progeny were reduced in appressorial penetration and lesion formation on rice leaves, the 12 hygromycin-sensitive progeny all were phenotypically similar to Guy11. These data indicated that the hygromycin resistance cosegregated with the reduced virulence of gas1 mutants. We also transformed pCX16 containing the full-length GAS1 gene (Figure 3) into gas1 mutant BC46 by cotransformation with pAC905. Among 24 zeocin-resistant transformants screened by PCR with primers C1F and C1R, four with the GAS1 gene integrated ectopically were identified and confirmed further by DNA gel blot analysis (data not shown). These four complemented GAS1 transformants were as virulent as Guy11. Thus, reintroduction of the wild-type GAS1 allele restored the appressorial penetration and lesion development defects in gas1 mutants.
gas2 Mutants Have Phenotypes Similar to Those of gas1 Mutants
The gas2 mutants also were normal in vegetative growth and sexual and asexual reproduction, but they were reduced in appressorial penetration and lesion formation (Table 1, Figure 4). On average, the gas2 mutants usually produced 10 lesions on the 5-cm leaf tip, an 77% reduction compared with Guy11 under the same conditions (Table 1). Morphologically, gas1 and gas2 mutants were not distinguishable. Twenty-six progeny were isolated from a cross between BE55 and 2539. Reduction in lesion formation was observed in all 10 hygromycin-resistant progeny but not in the 16 hygromycin-sensitive progeny (data not shown). Thus, the phenotypes observed in gas2 mutants were associated directly with the deletion of GAS2. We also generated gas1 deletion mutants in strain 70-15 and found that they had phenotypes similar to those of gas1 mutants in Guy11 (data not shown), indicating that the function of GAS2 is not strain specific.
gas1 gas2 Double Mutants Have Phenotypes Similar to Those of gas1 or gas2 Mutants To our surprise, the reduction in the appressoria penetration and lesion formation of gas1 gas2 double mutants was similar to that of the gas2 or gas1 mutants (Table 1). We repeated the infection and appressorial penetration assays several times and observed no significant difference between gas2 and gas1 gas2 double mutants. It is likely that the deletion of both GAS1 and GAS2 did not have any additive effect in gas1 gas2 double mutants.
Both GAS1 and GAS2 Localize to Cytoplasm in the Appressoria
We also examined conidiophores, young conidia, perithecia, and asci produced by GC22 and GE19 and did not observe any GFP signals. These data indicate that the Gas1-GFP and Gas2-GFP fusion proteins were expressed specifically in appressoria. The GFP fusion constructs pCX13 and pCX14 also were transformed into the gas1 mutant BC46 and the gas2 mutant BE55, respectively. MCX13 and MCX14 were two resulting transformants that were confirmed by DNA gel blot analysis to contain a single-copy integration of transforming vector pCX13 or pCX14. Both MCX13 and MCX14 were as efficient as Guy11 in appressorial penetration and lesion formation (data not shown), indicating that the Gas1-GFP and Gas2-GFP fusion proteins function normally in Magnaporthe. When examined by epifluorescence microscopy, the majority of appressoria expressing Gas1-GFP and Gas2-GFP fusion proteins had green fluorescence in the cytoplasm but not in the cell wall. We further confirmed these observations by examining the localization of GFP fusion proteins under confocal microscopy. Appressoria formed by both GE19 and GC22 had uneven fluorescent peripheries and strong green fluorescence in the cytoplasm (Figure 7B), indicating that Gas1-GFP and Gas2-GFP fusion proteins were localized in the cytoplasm but not in the cell wall or cytoplasm membrane. These GFP fusion proteins were not distributed evenly in the cytoplasm, however, and there were certain areas in the appressorium cytoplasm, possibly the lipid or glycogen bodies and nucleus, that had no detectable green fluorescence. Close examination indicated that the cellular localizations of Gas1 and Gas2 were slightly different. Although Gas2-GFP fusion proteins were distributed somewhat evenly throughout the cytoplasm, Gas1 proteins were localized preferentially in the vacuole in appressoria (Figure 7B). Interestingly, not all of the appressoria produced by transformants GE19 or GC22 displayed green fluorescence (Figure 7A). We then transformed pCX35 into GC22 and isolated transformants that contained GAS1-GFP and GAS2–yellow fluorescent protein (YFP) fusion constructs. In DT2, one of the transformants expressing both GAS1-GFP and GAS2-YFP, growth and virulence were normal (data not shown). However, even in DT2, there was always a small portion of appressoria that displayed no detectable fluorescence under epifluorescence microscopy (Figure 8). It is possible that the fluorescent fusion proteins were not expressed or were expressed in these appressoria at a level lower than the detection limit of the epifluorescence microscopy.
We also transformed pCX27, a GAS2-GFP fusion construct with the 3' untranslated region sequence of GAS2, into Guy11. In MCX27 and other transformants expressing this GAS2-GFP-TGAS2 construct, we also observed variation of the GFP signal among appressoria, and some appressoria had no detectable GFP signal at all (data not shown). No obvious difference in GFP signals was observed between appressoria formed by MCX27 and GE19 (Figure 7), indicating that the 3' untranslated region of GAS2 has no significant effect on the accumulation and localization of Gas2-GFP fusion proteins.
Expression Patterns of GAS1 and GAS2 Are Different during Appressorium Formation
At 6 h, >40% of appressoria formed by GE19 had strong green fluorescence. When examined from 12 to 48 h, the majority of appressoria (>80%) had strong green fluorescence. The Gas2-GFP fusion protein appeared to be more stable than Gas1-GFP, because 45% of GE19 appressoria remained fluorescent after 4 days of incubation. Interestingly, appressoria formed by transformants expressing GAS2-GFP usually had stronger fluorescence than those formed by transformants expressing GAS1-GFP (Figure 7) at any time points examined (data not shown), indicating that the expression level of GAS2-GFP was higher than that of GAS1-GFP in appressoria. To determine whether GAS1 and GAS2 can complement each other, we introduced pCX14 into BC46 and pCX13 into BE55. The resulting transformants had phenotypes similar to those of the original gas1 and gas2 mutants BC46 and BE55. No cross-complementation effect on appressorial penetration or lesion development was observed in gas1 and gas2 mutants transformed with pCX14 and pCX13, respectively. It is likely that GAS1 and GAS2 play some overlapping but different functions at different stages of appressorium formation.
GAS1 and GAS2 Are Not Expressed in Infectious Hyphae In some but not all primary infectious hyphae or pegs that penetrated into onion epidermal cells, weak fluorescence was observed (Figure 8), suggesting that the fluorescent fusion proteins accumulated in appressoria may be transported into the primary infectious hyphae or that the fusion constructs were expressed transiently at this penetration stage. Interestingly, many appressoria that failed to penetrate remained fluorescent, but none of the appressoria that produced infectious hyphae had any detectable fluorescence (Figure 8). It is likely that Gas1 and Gas2 proteins are functional only in appressoria and possibly are involved in penetration peg and primary infectious hyphae development. After penetration, these fusion proteins are either degraded rapidly or transported into penetration pegs. Data from these assays are consistent with our earlier observation that GAS1 and GAS2 were not detectable by RNA gel blot analysis or reverse transcriptase–mediated PCR with RNAs isolated from rice leaves collected at 60 h or 5 days after inoculation.
To further characterize the PMK1 MAP kinase pathway in Magnaporthe, two genes identified in a subtraction library enriched for genes regulated by PMK1 were characterized functionally in this study. Both GAS1 and GAS2 are highly expressed during appressorium formation and rich in Ala and Gly. Interestingly, there is a putative protein kinase C phosphorylation site (Figure 1) that is well conserved among GAS1, GAS2, and their homologs from other fungi. In Magnaporthe, pharmacological studies had indicated that protein kinase C may be involved in appressorium formation (Eckhard et al., 1998 ).
GAS1 or GAS2 or both are dispensable for mycelial growth, conidiation, and sexual reproduction, but they are important for appressorial penetration and lesion development. In Magnaporthe, several genes, such as MPG1, PTH11, and ABC1, are dispensable for vegetative growth and sexual or asexual reproduction but play important roles in appressorium formation and plant infection. Mutants disrupted in MPG1, a hydrophobin gene, were reduced by 74 and 80% in appressorium formation and lesion development, respectively (Talbot et al., 1993 Because the expression pattern and cellular localization of the Gas1-GFP and Gas2-GFP fusion proteins are slightly different, it is reasonable to assume that GAS1 and GAS2 may have different functions during appressorial penetration and infectious hyphal growth. Surprisingly, the gas1 gas2 double mutants have phenotypes similar to those of gas2 or gas1 mutants. No additive effect of gas1 and gas2 deletions was observed in the gas1 gas2 double mutants. One possible explanation for this finding is that GAS1 and GAS2 function sequentially in the penetration and lesion development processes. Mutants deleted in either the GAS1 or GAS2 gene will block the infection process to a level similar to that of the gas1 gas2 double mutants. The other possibility is that GAS1 and GAS2 may interact with each other and form a complex that is required for efficient penetration and lesion development. However, the Magnaporthe genome may contain additional GAS1 or GAS2 homolog(s) that can compensate partially for the deletion of GAS1 or GAS2. In E. graminis, two isoforms of Egh16H1 (A and B) that differ in only a few amino acid residues have been isolated as genes expressed in early infection stages. The exact biological function of gEgh16 and Egh16H1 isoforms in E. graminis is not clear.
Both GAS1 and GAS2 are expressed specifically in appressoria and play important roles in appressorium penetration. A few fungal genes are known to be expressed specifically in appressoria and are critical for plant infection. One of them is PLS1 in Magnaporthe, which encodes a transmembrane protein expressed only in appressoria, as determined by GFP-tagging assays (Clergeot et al., 2001
Two other Magnaporthe genes were reported to be expressed specifically in appressoria but were not characterized functionally (Lee and Dean, 1993
Both Gas1-GFP and Gas2-GFP fusion proteins localized to the cytoplasm in appressoria. When analyzed with various programs available on the World Wide Web for extracellular or subcellular localization, no consistent predictions were obtained for either Gas1 or Gas2. Interestingly, putative signal peptides at the N termini of Gas1 and Gas2 were identified by SignalP version 2.0 (Nielsen et al., 1997 We were unable to observe green fluorescence in the surrounding areas of either fluorescent or nonfluorescent appressoria formed by GE19, GC22, or DT2 (Figures 7 and 8). After removing appressoria by rubbing with gloved fingers, no green fluorescence was observed in the appressorial mucilage left behind (data not shown). Most likely, the Gas1 and Gas2 fusion proteins were not secreted from these appressoria. We were unable to detect green fluorescence in infectious hyphae or the transcript of GAS1 or GAS2 in infected rice leaves, indicating that these two genes were not expressed in infectious hyphae. However, it remains possible that the fluorescent fusion proteins may be expressed and secreted into plant cells at a level lower than the detection limit. Plant cells penetrated by Magnaporthe usually accumulate autofluorescent materials as part of the defense response, making it impossible to detect small amounts of secreted fluorescent fusion proteins. It may be necessary to isolate extracellular proteins secreted by Magnaporthe and analyze them by protein gel blot analysis.
In the GFP-tagging experiments with GAS1 and GAS2, there were always some normally shaped appressoria without any detectable green fluorescence. This is similar to what has been reported in transformants expressing the Pls1-GFP fusion protein, which had strong fluorescence only in 80% of appressoria (Clergeot et al., 2001
The expression of GAS1 and GAS2 may be regulated developmentally in these processes. The variation in GFP signal among appressoria may reflect different developmental stages of the appressoria we observed. MST12 is one of the transcription factors regulated by PMK1 to control appressorial penetration and infectious hyphae growth (Park et al., 2002
Strains and Culture Conditions Wild-type Magnaporthe grisea strains and various transformants generated in this study (Table 3) were cultured at 25°C on oatmeal agar plates or V8 juice agar under fluorescent light to induce conidiation (Xu and Hamer, 1996 ; Choi and Dean, 1997). Mycelia collected from 2-day-old 5 x YEG (5 mg/ml yeast extract and 10 mg/ml glucose; Zheng et al., 2000) cultures shaken at 150 rpm at room temperature were used for the isolation of fungal DNA and protoplasts. Genetic crosses and isolation of progeny were performed as described (Xu and Hamer, 1996). The mating type of each progeny was determined by mating tests and PCR with the MAT1-1–specific primers MATA1 (5'-AGCCTCATCAACGGCAA-3') and MATA5 (5'-GGCACGAACATGCGATG-3') and the MAT1-2–specific primers MATB15 (5'-CTCAATCTCCGTAGTAG-3') and MATB16 (5'-ACAGCAGTATAGCCTAC-3').
Molecular Manipulations with DNA and RNA Fungal DNAs were extracted using the cetyl-trimethyl-ammonium bromide protocol (Xu and Hamer, 1996 ). Plasmid DNAs were isolated with Qiagen plasmid preparation kits (Valencia, CA) and sequenced with the ABI Prism BigDye Terminator cycle sequencing kit (PE Applied Biosystems, Foster City, CA). Standard molecular biology procedures were followed for RNA and DNA gel blot analyses and enzymatic manipulations with DNAs and RNAs (Sambrook et al., 1989). Homolog searches of DNA/protein sequence databases were performed with the BLAST programs (Altschul et al., 1997). Amino acid sequence comparisons and alignments were made with the BESTFIT, PILEUP, T-COFFEE, and BOXSHADE programs in the GCG Wisconsin software package (Accelrys, San Diego, CA).
Construction of a Subtraction Library Enriched for Genes Regulated by PMK1
Construction and Differential Screening of an Appressorium-Stage cDNA Library
Construction of the GAS1 and GAS2 Gene Replacement Vectors A similar approach was used to construct the GAS2 gene replacement vector pCX3. The 0.6-kb upstream sequence amplified with primers E3F (5'-CAATGTGCAAGAGAGTCAGCA-3') and E2R (5'-AGTGCCATCGATGGCGGTCA-3') was cloned into pGEM-T Easy as pMBEA. The hph cassette released from pCB1003 and the downstream flanking sequence of GAS2 amplified with primers E2F (5'-GAGGATGACGAGTAGAGTGT-3') and E4R (5'-GAGTGTTGAGAG-TTGAGACA-3') were cloned between the KpnI and BamHI sites and the BamHI and BstXI sites on pMBEA, respectively, to generate pCX3.
Isolation of gas1 and gas2 Deletion Mutants
All putative gene replacement mutants identified in PCR screens were confirmed further by DNA gel blot analyses. For complementation assays, a 4.2-kb NcoI fragment containing the full-length GAS1 gene (Figure 3) was cloned into pGEM-5Zf (Promega) and cotransformed into the gas1 mutant BC46 with the bleomycin-resistant vector pAC905 (Zheng et al., 2000
Assaying Germination, Appressorium Formation, and Penetration
Plant Infection Assays
Construction and Expression of Gas1–Green Fluorescent Protein and Gas2–Green Fluorescent Protein Fusion Proteins
We also cloned the PCR product amplified by EU2F and EU2R into pEYFP (Clontech) as pCX26. The GAS2–yellow fluorescent protein (YFP) fusion construct was released from pCX26 with HindIII and NotI digestion and cloned into pCB1004 (Carroll et al., 1994 pCX13 and pCX14 were introduced individually into Guy11 by cotransformation with pAC905. Zeocin-resistant transformants were screened by PCR with primers GP1F (5'-CATCCTGGTCGAGCT-GGA-3') and GP1R (5'-CTTGTACAGCTCGTCCATG-3') and confirmed by DNA gel blot analysis. The expression of GFP was examined on both glass cover slips (Fisher Scientific) and onion epidermis with a Nikon Eclipse E800 epifluorescence microscope (Tokyo, Japan). The Chroma Endow GFP filter on this microscope is suitable to detect both GFP and YFP but not to distinguish them. The subcellular GFP localization also was examined with a Bio-Rad MRC 1024 UV/Vis System confocal laser scanning microscopy (Hercules, CA) on infected barley leaves and glass cover slips. Transformants expressing both GAS1-GFP and GAS2-YFP fusion constructs were generated by transforming pCX35 into GC22, a Guy11 transformant carrying GAS1-GFP. Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
We thank Larry Dunkle, Stephen Goodwin, and Charles Woloshuk at Purdue University for critical reading of the manuscript. We also thank Richard Howard at Dupont for useful suggestions on the GFP-tagging experiments and Mark Farman at the University of Kentucky for communicating unpublished data. This work was supported by U.S. Department of Agriculture National Research Initiative Grant 2001-35319-09924 to J.-R.X. and by grants from the National Science Foundation to R.A.D.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003426. Received March 25, 2002; accepted June 14, 2002.
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Abstract
INTRODUCTION
, 550 white colonies were picked and preserved individually as the subtraction library. 
