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First published online April 22, 2008; 10.1105/tpc.107.056895

The Plant Cell 20:824-826 (2008)
© 2008 American Society of Plant Biologists

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LETTER TO THE EDITOR

The coi1-16 Mutant Harbors a Second Site Mutation Rendering PEN2 Nonfunctional

Lore Westphal, Dierk Scheel and Sabine Rosahl

Department of Stress and
Developmental Biology
Leibniz Institute of Plant Biochemistry
D-06120 Halle (Saale), Germany

srosahl{at}ipb-halle.de

Coronatine is a phytotoxin produced by several pathovars of Psedudomonas syringae that acts as a mimic of methyl jasmonate in plants. Due to the importance of jasmonic acid and its derivatives in responses of plants to biotic and abiotic stress, the Arabidopsis thaliana coronatine-insensitive mutant coi1 is widely used in the plant community. Ellis and Turner (2002)Go described a conditional fertile coi1 allele, coi1-16, that is being used in a number of laboratories. We have discovered that the coi1-16 mutant (but not the original coi1-1) carries an additional mutation, which might also influence the responses usually studied with coi1 (i.e., pathogen defense responses). This additional mutation lies in the PENETRATION2 (PEN2) gene, which was identified as a gene required for nonhost resistance of Arabidopsis against barley powdery mildew (Lipka et al., 2005Go).

Nonhost resistance describes the ability of all members of a plant species to successfully prevent colonization by any member of a given pathogen species. Nonadapted pathogens are recognized by the nonhost plant via pathogen-associated molecular patterns, which leads to the activation of defense responses (Nürnberger and Lipka, 2005Go). Mutant screens have identified genes important for nonhost resistance of Arabidopsis against the nonadapted barley powdery mildew fungus Blumeria graminis f. sp hordei (Bgh). PEN1 encodes a SNARE-syntaxin that is involved in vesicle transport to the site of attempted penetration (Collins et al., 2003Go; Kwon et al., 2008Go). A defect in the PEN2 gene, encoding a glycoside hydrolase, leads to a loss of penetration resistance (Lipka et al., 2005Go), as does a mutation in PEN3 encoding an ABC transporter (Stein et al., 2006Go). The PEN2 protein is a member of the Arabidopsis family 1 glycoside hydrolases. Although the catalytic activity of PEN2 has yet to be shown, the substitution of a Glu to Asp at the putative active site renders the protein unable to complement the pen2 mutant, suggesting that catalytic activity is required for PEN2 function (Lipka et al., 2005Go).

Apparently, the PEN proteins collectively comprise a set of pre-invasion defense responses activated in nonhost plants, which restrict pathogen growth at the cell periphery. Once this pre-invasion defense is compromised, additional layers of defense responses are able to restrict further spread of the pathogen, since all three pen mutants are still resistant to Bgh. Postinvasion defense responses against Bgh require functional PAD4, EDS1, and SAG101 genes (Lipka et al., 2005Go). On the pen2 pad4 sag101 triple mutant plant, Bgh is able to form conidiospores, suggesting a breakdown of nonhost resistance (Lipka et al., 2005Go).

The oomycete Phytophthora infestans, the causal agent of late blight disease of potato, is not able to infect Arabidopsis (Kamoun, 2001Go). This nonhost–pathogen interaction is characterized by the unsuccessful attempt of the oomycete to penetrate epidermal cells. Cessation of pathogen growth correlates with massive cell wall depositions in epidermal cells (Lipka et al., 2005Go). Phenotypically, no major symptoms can be detected in gl1, a trichomeless mutant that represents the wild type to pen2-1 (Figure 1A ). The pen2 and pen3 mutants, but not pen1, are compromised in penetration resistance against P. infestans (Lipka et al., 2005Go). pen2 plants react with visible necrosis formation (Figure 1A), and trypan blue staining shows an increased number of dead cells in pen2 compared with gl1 (Figure 1B). Postinvasion resistance against P. infestans does not require the same components as that against Bgh, since P. infestans does not show enhanced growth on pen2 pad4 sag101 triple mutant plants (data not shown).


Figure 1
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  		Figure 1. Mutant Phenotypes.

(A) Phenotype of gl1, pen2-1, coi1-16, and coi1-1 plants after infection with P. infestans. Plants were infected by drop inoculation with a zoospore solution of P. infestans (5 x 105 spores/mL). Photos were taken 3 d after inoculation.

(B) Visualization of cell death by trypan blue staining. Leaves of gl1, pen2-1, coi1-16, and coi1-1 plants were infected with a zoospore solution of P. infestans (5 x 105 spores/mL) and subjected to trypan blue staining 3 d after infection.

 
To identify additional genes or pathways required for nonhost resistance against P. infestans, mutants compromised in pathogen responses as well as salicylic acid, jasmonic acid (JA), and ethylene signaling were analyzed. Among these, coi1-16 shows a clear necrosis phenotype upon drop inoculation of a P. infestans zoospore solution (Figure 1A). The phenotype observed for coi1-16 is similar in extent to that observed on the pen2 mutant, and the intensity of trypan blue staining in samples from coi1-16 plants, similar to that of pen2 plants, is greater than that observed for gl1 plants (Figure 1B). These results suggested that JA signaling is required for nonhost resistance of Arabidopsis against P. infestans.

COI1 encodes an F-box protein that is required for the activation of JA-dependent responses. Upon increases in JA levels, COI1 is responsible for the specific degradation of jasmonate ZIM domain proteins, which act as negative regulators of JA-dependent gene expression by binding to the transcriptional activator MYC2/JIN1 (Chini et al., 2007Go; Thines et al., 2007Go). The mutant coi1-1 is male sterile and shows increased susceptibility to pathogens and herbivory (Stintzi et al., 2001Go). The conditional mutant coi1-16 was isolated from a mutant screen for methyl jasmonate–insensitive reporter gene expression. coi1-16 is fertile at temperatures below 20°C; however, root growth inhibition and JA-responsive promoter activity are not restored at lower temperatures (Ellis and Turner, 2002Go).

To analyze putative additive effects, we crossed the pen2-1 and coi1-16 mutants. Surprisingly, no complementation of the pen2-1 hypersensitive response (HR) phenotype occurred in the F1 generation (Table 1 ). Moreover, there was no segregation of the HR phenotype in the F2 generation (Table 1). This suggested either that coi1-16 carries a defective PEN2 gene, which would not be able to complement the pen2-1 mutant, or that pen2-1 contains a mutated COI1 gene. However, the pen2-1 mutant is not impaired in male fertility, as would be expected for a plant carrying a defective COI1 gene (Xie et al., 1998Go). Moreover, we observed segregation of the coi1 phenotype (i.e., male sterility) in the F2 generation at nonpermissive temperatures (Table 1). In contrast with coi1-16, coi1-1 does not display the HR phenotype after infection with P. infestans (Figure 1).


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  		Table 1. Segregation Analysis of the Cross between coi1-16 and pen2-1

 
Therefore, we cloned and sequenced the PEN2 gene from the coi1-16 mutant and found that it contains a G-to-A nucleotide exchange corresponding to position 449 of the cDNA. This mutation, subsequently called pen2-4, leads to an amino acid exchange from Gly to Asp at position 150. In addition, the G-to-A transition results in the loss of an AciI restriction site (Figures 2A and 2B ). To rule out the possibility that the wild type to coi1-16 already carries this mutation, gl1 plants were analyzed. The absence of the HR phenotype in gl1 plants correlated with the presence of the AciI restriction site, suggesting that gl1 does not contain the pen2-4 mutant allele (Figure 2B). Moreover, the PEN2 fragment amplified from genomic DNA of coi1-1 plants carried the AciI restriction site and yielded restriction fragments of the same size as did gl1 and pen2-1 (Figure 2B).


Figure 2
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  		Figure 2. Structure of PEN2 and PEN2-4 Genes.

(A) Structure of the PEN2 gene and location of the mutation in exon 5 in the pen2-4 gene from coi1-16 (marked by an asterisk). The G-to-A transition results in the elimination of an AciI site. Below are shown the positions of the AciI restriction sites in a 706-bp fragment amplified from PEN2, pen2-1, and pen2-4.

(B) Restriction of the 706-bp fragment from PEN2 (amplified using the primers 5'-AAACGTTGCCGTTGATTTCT-3'and 5'-CAGCAACACTAGCGCCATTA-3') from gl1, pen2-1, coi1-16, and coi1-1 plants with AciI. The letters "a" and "b" indicate two different lines of coi1-16.

 
To address possible causes of the pen2 phenotype of coi1-16, PEN2-4 protein levels in unchallenged coi1-16 plants were analyzed using PEN2 antiserum (Lipka et al., 2005Go). PEN2 protein is not detectable in pen2-1 plants compared with significant levels of PEN2 in gl1 plants (Figure 3 ). We found highly reduced amounts of PEN2-4 protein in coi1-16 plants, whereas coi1-1 contains PEN2 to similar levels as gl1 (Figure 3). These results suggest that the protein encoded by the pen2-4 gene of coi1-16 is unstable, and the inability of PEN2-4 to accumulate to high levels would explain the pen2 phenotype of coi1-16.


Figure 3
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  		Figure 3. Determination of PEN2 Protein Levels in gl1, pen2-1, coi1-16, and coi1-1 Plants.

Proteins were extracted from leaves of untreated plants and subjected to immunoblot analyses using PEN2 antiserum ({alpha}PEN2; Lipka et al., 2005Go). Filters were stained with amido black to visualize equal loading. LSU, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

 
These analyses show that coi1-16 carries a mutant allele of PEN2, which we have named pen2-4. In contrast with the knockout pen2 alleles pen2-1, pen2-2, and pen2-3 (Lipka et al., 2005Go), the pen2-4 allele encodes a protein with highly reduced stability. PEN2 is required for penetration resistance against nonadapted pathogens and, importantly, is also involved in defense against host pathogens, such as Plectosphaerella cucumerina and Pythium irregulare (Lipka et al., 2005Go; Adie et al., 2007Go). Therefore, studies with pathogens using coi1-16 should be evaluated carefully because the mutation in pen2, rather than the one in coi1, might be responsible for alterations in pathogen-related phenotypes.

Our findings emphasize the importance of performing multiple backcrosses to minimize the risk of phenotypic misinterpretation due to a second site mutation. However, for closely linked mutations, even repeated backcrossing cannot ensure successful separation. Therefore, complementation experiments or resequencing of ethyl methanesulfonate mutants are required to conclusively ascribe a mutant phenotype to the loss of a specific gene function.

Acknowledgments

The pen2-1 and coi1-16 mutants were kindly provided by P. Schulze-Lefert (Max Planck Institute for Plant Breeding Research, Cologne, Germany) and J. Turner (University of East Anglia, UK), respectively. Y. He and J. Dangl (University of North Carolina) are gratefully acknowledged for helpful discussions. We also thank V. Lipka (Sainsbury Laboratory, John Innes Center, Norwich, UK) and P. Schulze-Lefert for the PEN2 antiserum, M. Häuβler (Institute of Plant Biochemistry) for technical assistance, and K. Rejall (Institute of Plant Biochemistry) for taking care of the plants. This work was funded by the Deutsche Forschungsgemeinschaft (SPP 1212, "Microbial Reprogramming of Plant Cell Development").

Footnotes

www.plantcell.org/cgi/doi/10.1105/tpc.107.056895

REFERENCES

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Xie, D.X., Feys, B.F., James, S., Nieto-Rostro, M., and Turner, J.G. (1998). COI1: An Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280: 1091–1094.[Abstract/Free Full Text]





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