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PLANTS INTERACTING WITH OTHER ORGANISMS

Premature Leaf Senescence Modulated by the Arabidopsis PHYTOALEXIN DEFICIENT4 Gene Is Associated with Defense against the Phloem-Feeding Green Peach Aphid^1,[W]

Division of Biology and the Molecular Cellular and Developmental Biology Program (V.P., C.K., J.S.), and Department of Entomology (J.R.), Kansas State University, Manhattan, Kansas 66506–4901

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Aphids, which are phloem-feeding insects, cause extensive loss of plant productivity and are vectors of plant viruses. Aphid feeding causes changes in resource allocation in the host, resulting in an increase in flow of nutrients to the insect-infested tissue. We hypothesized that leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf, could be utilized by plants to limit aphid growth. Using Arabidopsis (Arabidopsis thaliana) and green peach aphid (GPA; Myzus persicae Sulzer), we found that GPA feeding induced premature chlorosis and cell death, and increased the expression of SENESCENCE ASSOCIATED GENES (SAGs), all hallmarks of leaf senescence. Hypersenescence was accompanied by enhanced resistance against GPA in the Arabidopsis constitutive expresser of PR genes5 and suppressor of SA insensitivity2 mutant plants. In contrast, resistance against GPA was compromised in the phytoalexin deficient4 (pad4) mutant plant. The PAD4 gene, which is expressed at elevated level in response to GPA feeding, modulates the GPA feeding-induced leaf senescence. In comparison to the wild-type plant, GPA feeding-induced chlorophyll loss, cell death, and SAG expression were delayed in the pad4 mutant. Although PAD4 is associated with camalexin synthesis and salicylic acid (SA) signaling, camalexin and SA signaling are not important for restricting GPA growth; growth of GPA on the camalexin-biosynthesis mutant, pad3, and the SA deficient2 and NahG plants and the SA-signaling mutant, nonexpresser of PR genes1, were comparable to that on the wild-type plant. Our results suggest that PAD4 modulates the activation of senescence in the aphid-infested leaves, which contributes to basal resistance to GPA.

Our knowledge of plant defense against insects is based largely on studies involving chewing insects. Far less is known about plant defense mechanisms against aphids and other piercing/sucking insects (Walling, 2000). Due to their feeding behavior, unlike the chewing insects, aphids do not cause extensive wounding to the plant host, suggesting that plant response to phloem-feeding insects may differ from that to the chewing insects (Walling, 2000). A few studies have identified plant genes associated with defense against aphids. In tomato (Lycopersicon esculentum), the Mi1.2 gene, which encodes a nucleotide-binding site, Leu-rich repeat protein, mediates gene-for-gene resistance against certain biotypes of the potato (Solanum tuberosum) aphid Macrosiphum euphorbiae (Rossi et al., 1998; Vos et al., 1998). Similarly, the apple Sd₁ gene confers resistance to two biotypes but not a third biotype of the rosy leaf-curling aphid, Dysaphis devecta (Roche et al., 1997), while the lettuce (Lactuca sativa) Nr gene confers resistance to a single aphid species, Nasonovia ribisnigri (van Helden et al., 1993). Comparable to the function of Resistance genes in plant response to pathogen infection (Bent, 1996; Hammond-Kosack and Jones, 1996), interaction of aphid-generated or -derived signal with a Resistance gene-encoded protein may presumably activate a signal transduction pathway(s) that confers expression of an appropriate defense response(s) against the aphid. Expression of the salicylic acid (SA)-inducible Pathogenesis-Related genes, which are involved in plant defense against pathogens (Dempsey et al., 1999; Shah and Klessig, 1999), are also induced by aphid feeding (Fidantsef et al., 1999; Moran and Thompson, 2001; Moran et al., 2002; Zhu-Salzman et al., 2004). In addition, expression of the SA DEFICIENT2 (SID2) gene, which encodes an enzyme involved in SA biosynthesis, the ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) gene, which is required for SA biosynthesis, and the PHYTOALEXIN DEFICIENT4 (PAD4) gene, which modulates SA signaling, are induced in Arabidopsis (Arabidopsis thaliana) in response to GPA feeding (V. Pegadaraju and J. Shah, unpublished data). However, loss-of-function mutations in the EDS5 gene and the Arabidopsis NONEXPRESSER OF PR GENES1 (NPR1) gene, which is required for SA signaling, do not compromise resistance to GPA (Moran and Thompson, 2001), suggesting that SA accumulation and signaling may not have an important role in Arabidopsis defense against GPA. Recent studies suggest a role for oxylipin signaling in plant defense against aphids. For example, greenbug feeding transiently induced the expression of jasmonic acid (JA)-induced genes in sorghum (Sorghum bicolor; Zhu-Salzman et al., 2004). Similarly, the expression of JA-responsive genes was induced in Arabidopsis infested with GPA (Moran et al., 2002). Moreover, the Arabidopsis coronatine-insensitive1 mutant, which is compromised in oxylipin signaling, supported more growth of GPA than the wild-type plant did (Ellis et al., 2002). In support of a role for JA in plant defense against aphids, the application of methyl jasmonate caused a significant reduction in greenbug infestation in sorghum (Zhu-Salzman et al., 2004).

Aphid infestation causes changes in resource allocation in the host plant; flow of nutrients to the insect-infested tissue is increased due to the creation of a strong sink in the aphid-infested organ (Mittler and Sylvester, 1961; Dixon, 1998; Girousse et al., 2005). However, flow of nutrients to the natural resource-demanding sinks, like the primary growth zones, is reduced. In fact, aphid infestation converts the uninfested natural sink tissues into source tissues (Girousse et al., 2005). From the aphid's perspective, an increase in the sink level of a tissue amounts to improved nutrient availability. Gene expression studies confirm that aphid infestation alters expression of plant genes that are potentially involved in the conversion of the feeding site into metabolic sinks. GPA feeding induced expression of the Arabidopsis STP4 gene, which encodes a monosaccharide H⁺ symporter (Moran and Thompson, 2001; Moran et al., 2002), and another gene that encodes an extracellular acidic invertase (V. Pegadaraju and J. Shah, unpublished data). STP proteins along with invertases increase the import and metabolism of carbohydrates into resource-demanding organs (Buttner et al., 2000).

Senescence is the terminal phase in leaf development that involves a programmed disassembly and degradation of cellular components (Lim et al., 2003; Thomas et al., 2003; Yoshida, 2003). The resultant products of senescence are remobilized to assimilate demanding sink organs. In Arabidopsis, leaf senescence is characterized by chlorophyll loss, elevated level of expression of the SENESCENCE ASSOCIATED GENES (SAGs), and eventually cell death (Lim et al., 2003; Yoshida, 2003). Premature senescence of the aphid-infested leaf could oppose the ability of aphids to redirect the flow of resources to the insect-infested leaves. We therefore hypothesized that leaf senescence may be utilized as a defense mechanism by plants to limit aphid growth. Here we show that GPA feeding induces premature leaf senescence in Arabidopsis. Furthermore, in comparison to the wild-type Arabidopsis plant, GPA growth is lower on mutants that exhibit hypersenescence. In contrast, a delay in the activation of GPA feeding-induced leaf senescence is accompanied by increased growth of GPA in the pad4 mutant plant. These data implicate a role for PAD4-modulated leaf senescence in basal resistance to GPA.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

GPA Feeding Activates Leaf Senescence in Arabidopsis

We first examined whether GPA feeding results in chlorophyll loss and cell death, two features of leaf senescence in Arabidopsis. In comparison to leaves from uninfested plants (Supplemental Fig. 1), as shown in Figure 1A, GPA feeding resulted in leaf chlorosis in wild-type Arabidopsis. In comparison to the uninfested wild-type plants, by 2 d postinfestation (dpi) GPA-infested leaves from the wild-type plant had lost 40% of their chlorophyll (Fig. 1B). In addition, microscopy of trypan blue-stained leaves revealed the presence of dead cells in the GPA-infested leaves from wild-type plants at 2 dpi (Fig. 1C). Senescence requires the de novo expression of genes (Gan and Amasino, 1997; Lim et al., 2003; Yoshida, 2003). To confirm that the chlorophyll loss and cell death observed in GPA-infested leaves is part of a plant response to aphid feeding and not a consequence of cell damage caused by insect probing/feeding, we examined expression of the SAG genes in the GPA-infested leaves. Expression of the SAG13, SAG21, and SAG27 genes was induced as early as 12 h postinfestation (hpi) in GPA-infested leaves from wild-type plants (Fig. 1D). Elevated expression of the SAG12 gene correlates with the manifestation of age-dependent senescence but not senescence induced by environmental stressors (Gan and Amasino, 1997; Lim et al., 2003; Yoshida, 2003). SAG12 expression is first evident when the senescing organs undergo chlorosis (Weaver et al., 1998). However, in the GPA-infested leaves from the wild-type plant, SAG12 expression was undetectable even at 72 hpi (Fig. 1D), although chlorophyll loss was evident by 48 hpi (Fig. 1B). We therefore suggest that the senescence phenomenon observed in GPA-infested leaves of wild-type plants may differ from age-dependent senescence.

View larger version (33K): [in this window] [in a new window]   Figure 1. GPA feeding activates leaf senescence that is modulated by PAD4. A, Photograph of wild-type (WT) and pad4-1 leaves, 7 d after release of 15 GPA per plant. B, Relative chlorophyll content in GPA-infested leaves of wild-type and pad4-1 plants, 2 d after release of 15 insects per plant. The chlorophyll values in the GPA-infested wild-type and pad4-1 plants are relative to that in the corresponding uninfested wild-type and pad4-1 plants, which were assigned a value of 100. This experiment was done twice with similar results. C, Trypan blue staining of leaves from uninfested wild-type and pad4-1 plants and from GPA-infested wild-type and pad4-1 plants, 2 d after release of insects. The arrows point to the intensely stained dead cells. D, RT-PCR analysis of SAG12, SAG13, SAG21, SAG27, and ACT8 expression in leaves from uninfested wild-type and pad4-1 plants and leaves from GPA-infested wild-type and pad4-1 plants, 3, 6, 12, 24, 48, and 72 hpi by GPA. ACT8 expression provided a control for RT-PCR.

If a senescence-associated process influences Arabidopsis defense against GPA, then we expect that hypersenescence will be coupled with enhanced resistance against GPA. The Arabidopsis constitutive expresser of PR genes5 (cpr5)/hypersenescence1 mutant plant exhibits a hypersenescent phenotype; in comparison to the wild-type plant, the mutant plant has a lower content of chlorophyll, spontaneously undergoes cell death, and exhibits elevated basal levels of SAG gene expression (Fig. 2A; Bowling et al., 1997; Yoshida et al., 2002). We monitored aphid performance on the cpr5 mutant plant. In comparison to the wild-type plant, GPA counts were lower on the cpr5 mutant (Fig. 2B). Similarly, in comparison to the wild-type plant, GPA growth was reduced in the Arabidopsis suppressor of SA insensitivity2 (ssi2) mutant (Fig. 2C), which like cpr5 contains high basal levels of the SAG13, SAG21, and SAG27 transcripts (Fig. 2A) and exhibits spontaneous cell death (Shah et al., 2001). The cpr5 and ssi2 mutants are dwarfs and accumulate high levels of SA (Bowling et al., 1997; Shah et al., 2001), an important signaling molecule in plant defense to pathogens. However, GPA growth was comparable on the ssi2 single mutant and the SA-deficient ssi2 nahG plant (Fig. 2C), suggesting that a high level of SA is not important for the ssi2-conferred resistance to GPA. Aphid growth was also comparable between the wild-type plant and the Arabidopsis SA-deficient nahG (Fig. 2C) and sid2, and the SA-insensitive npr1 mutant plants (Fig. 3A; Moran and Thompson, 2001). In addition, GPA counts on the suppressor of npr1-1, constitutive 1 (snc1) mutant, which accumulates high levels of SA and is a dwarf (Zhang et al., 2003) like the cpr5 and ssi2 mutants, were comparable to those on the wild-type plant (Fig. 3B). Moreover, unlike the cpr5, ssi2, and ssi2 nahG plants, the SAG13, SAG21, and SAG27 genes were not expressed constitutively in the snc1 mutant (Fig. 2A; data not shown), thus supporting the hypothesis that a senescence-associated process, but not SA or dwarfing, is linked with basal resistance to the GPA.

View larger version (21K): [in this window] [in a new window]   Figure 2. Hypersenescence is accompanied by enhanced resistance to GPA in the cpr5 and ssi2 mutant plants. A, Left section: RT-PCR analysis of SAG13, SAG21, SAG27, and ACT8 expression in leaves of uninfested ecotype Nössen (WT No) and Columbia (WT Col) plants, and the ssi2 and cpr5 mutant plants. Right section: RT-PCR analysis of SAG13, SAG21, SAG27, and ACT8 expression in leaves of uninfested wild-type Col and the snc1 mutant plants. B, Comparison of GPA growth on the wild-type (ecotype Columbia) and cpr5 mutant plants, 2 d after release of 15 insects per plant. C, Comparison of GPA growth on the wild-type (ecotype Nössen), and the ssi2, ssi2 nahG, and nahG plants, 2 d after release of 15 insects per plant. These plants are in the ecotype Nössen background. In B and C, all values are the mean of 15 plants ±SE. Different letters above the bars indicate values that are different from each other with a confidence of 95% or greater with Student's t test.

View larger version (13K): [in this window] [in a new window]   Figure 3. SA does not have an important role in basal resistance to GPA. A, Comparison of GPA numbers on wild type and the SA-deficient sid2 and the SA-insensitive npr1-1 mutant plants, 2 d after release of 15 insects per plant. B, Comparison of GPA numbers on wild type and the SA-hyperaccumulating snc1 mutant plant, 2 d after release of 15 insects per plant. All values are the mean of 15 plants ±SE. Different letters above the bars indicate values that are different from each other with a confidence of 95% or greater with Student's t test.

In contrast to the hypersenescence mutants, cpr5 and ssi2, the GPA feeding-induced senescence is delayed in the Arabidopsis pad4-1 mutant plant. The GPA-infested pad4-1 mutant plant stayed green for longer than the GPA-infested wild-type plant (Fig. 1A). Measurements on chlorophyll content confirmed that, in comparison to the wild-type plants, the GPA feeding-induced chlorophyll loss was compromised in the pad4-1 mutant plant (Fig. 1B). Furthermore, the GPA feeding-induced expression of SAG13, SAG21, and SAG27 genes was delayed in the pad4-1 mutant (Fig. 1D). Unlike in the wild-type leaves, microscopic cell death was also not evident in the GPA-infested pad4-1 leaves at 2 dpi (Fig. 1C). In agreement with the involvement of PAD4 in Arabidopsis response to GPA, PAD4 expression was activated in the GPA-infested wild-type plants as early as 3 hpi and was maintained at high levels through 48 hpi (Fig. 4A). These results suggest that PAD4 modulates the activation of the GPA feeding-induced leaf senescence process in Arabidopsis.

View larger version (27K): [in this window] [in a new window]   Figure 4. PAD4 is involved in Arabidopsis defense against GPA. A, RT-PCR analysis of PAD4 and ACT8 expression in GPA-infested Arabidopsis leaves. RT-PCR was performed on RNA extracted 3, 6, 12, 24, and 48 hpi. RNA extracted from uninfested plants provided a negative control. ACT8 expression provided a control for RT-PCR. B, Comparison of GPA numbers on wild type and pad4-1 mutant (left section), and wild type and a transgenic line (pad4) that contains a T-DNA insertion within the PAD4 gene (right section), 2 d after release of 15 insects per plant. All values are the mean of aphid counts on 15 plants ±SE. C, Seed yield from uninfested and GPA-infested wild-type and pad4-1 mutant plants. All values are the mean of seed yield from 10 plants ±SE. Different letters above the bars indicate values that are different from each other with a confidence of 95% or greater with Student's t test. This experiment was done twice with similar results.

The PAD4 gene modulates SA signaling and synthesis of camalexin, an antimicrobial phytoalexin (Tsuji et al., 1992; Rogers et al., 1996; Zhou et al., 1998; Jirage et al.,1999). However, as shown above, SA accumulation and signaling are not critical for basal resistance to GPA. The PAD3 gene encodes a cytochrome P450 monooxygenase, a key enzyme in camalexin biosynthesis (Zhou et al., 1999). Like PAD4, expression of the PAD3 gene is induced in Arabidopsis leaves in response to GPA feeding (V. Pegadaraju and J. Shah, unpublished data). To ascertain if camalexin has a role in basal resistance to GPA, we compared aphid counts between the pad3-1 mutant and wild-type plant. A comparable number of aphids was present on the pad3-1 mutant and wild-type plant (Fig. 5), suggesting that camalexin is not important for basal resistance to GPA. Hence, the involvement of PAD4 in Arabidopsis defense against GPA is most likely independent of its role in SA signaling and camalexin biosynthesis.

View larger version (10K): [in this window] [in a new window]   Figure 5. Camalexin is not required for basal resistance to GPA. Comparison of GPA numbers on wild type and the camalexin-deficient pad3 mutant, 2 d after release of 15 insects per plant. All values are the mean of 15 plants ±SE. Different letters above the bars indicate values that are different from each other with a confidence of 95% or greater with Student's t test.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Flowers and developing seeds are natural sinks. In comparison to the uninfested wild-type plants, GPA feeding reduced seed set in the GPA-infested plants (Fig. 4C), confirming observations made by others (Mittler and Sylvester, 1961; Dixon, 1998; Girousse et al., 2005) that aphid feeding alters plant source-sink relationships. However, in comparison to the GPA-infested wild-type plant, seed set in GPA-infested pad4 mutant plant was further reduced (Fig. 4C), suggesting that the wild-type plant has countermeasures to limit the ability of the aphid to alter source-sink relationships. Our results suggest that PAD4 is a component of this counter mechanism. The influence of senescence-associated processes on aphid growth has also been observed in other plants. For example, premature senescence induced by a gall aphid was shown to correlate with the reduced performance of another aphid feeding on the same leaflet of Pistacia palaestina trees (Inbar et al., 1995). Furthermore, in barley (Hordeum vulgare), resistance to Russian wheat aphid was accompanied by the activation of cell death in the resistant cultivars (Belefant-Miller et al., 1994). Similarly, premature leaf senescence also benefits plants during drought stress. Nutrient remobilization associated with drought-induced leaf senescence allows the natural sink organs like the young leaves, fruits, and flowers to benefit from the nutrients accumulated during the life span of the prematurely senescing leaf (Munné-Bosch and Alegre, 2004).

Our data suggest that PAD4 modulates the GPA feeding-induced premature leaf senescence. Previously, the PAD4 gene was shown to influence the manifestation of hypersensitive response-like cell death in the Arabidopsis acd11 and lsd1 mutants, and the acd11-conferred constitutive high-level expression of SAG13 (Rustérucci et al., 2001; Brodersen et al., 2002), providing additional support for a role for PAD4 in activation of senescence-associated processes. PAD4 modulates camalexin synthesis and SA signaling in plant defense against pathogens (Glazebrook et al., 1997; Zhou et al., 1998; Jirage et al., 1999; Zhou et al., 1999). However, our studies of GPA performance on the camalexin biosynthesis mutant, pad3 (Fig. 5), suggest that camalexin does not have an important role in basal resistance to GPA. An earlier study in Arabidopsis found no correlation between the activation of SA signaling and basal resistance to GPA. GPA growth was comparable between the wild-type plant and the SA-insensitive npr1 and the SA-deficient eds5 mutant plants (Moran and Thompson, 2001). However, both NPR1 and EDS5 also participate in processes that are independent of SA (Pieterse et al., 2002; Nandi et al., 2005). Our experiments with the nahG plant (Fig. 2C), in which SA is degraded to catechol, the SA biosynthesis mutant sid2 (Fig. 3A), and the SA-hyperaccumulating snc1 mutant (Fig. 3B) plants, extend Moran and Thompson's (2001) study and confirm that SA does not have an important role in basal resistance to GPA. Hence, we propose that the participation of PAD4 in plant defense against GPA is independent of its involvement in camalexin synthesis and SA signaling. A similar association of PAD4 in the expression of Arabidopsis genes, which is independent of PAD4s involvement in SA signaling, was observed in a microarray gene expression study (Glazebrook et al., 2003). Moreover, unlike the involvement of PAD4 in SA signaling, which is dependent on the presence of a functional EDS1 gene (Feys et al., 2005), EDS1 is not important for basal resistance to GPA (V. Pegadaraju, J. Parker, and J. Shah, unpublished data), suggesting that the role of PAD4 in Arabidopsis-GPA interaction is independent of its interaction with EDS1. PAD4 protein localizes to the nucleus (Feys et al., 2005), where it may modulate the expression of genes or activity of proteins involved in the activation of this GPA-induced leaf senescence. Alternatively, since PAD4 protein can also be found in the cytosol (Feys et al., 2005), the involvement of PAD4 in Arabidopsis-GPA interaction may be due to its action in the cytosol.

Although premature senescence of aphid-infested leaves may appear as a consequence of removal of nutrients by the insect from the infested organ, our study demonstrates that PAD4-modulated leaf senescence in Arabidopsis contributes to defense against the generalist insect, GPA. These results have broader ramifications to agriculture because delayed senescence is one of the traits that is being selected to improve productivity in several crops (Ma and Dwyer, 1998; Ismail et al., 2000; Borrel et al., 2001; Mahalaxmi and Bidinger, 2002; Munné-Bosch and Alegre, 2004). Will delayed senescence in these crops result in lowered resistance to aphids and in parallel an increase in the spread of aphid-vectored viral diseases? Similarly, concerns have been raised about drought tolerance in plants with delayed senescence (Munné-Bosch and Alegre, 2004).

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant and Aphid Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants were grown in soil at 22°C in a growth chamber programmed for 14-h-light (100 µE m^–2 s^–1) and 10-h-dark cycle. Approximately 4-week-old Arabidopsis plants were used for all studies. A combination of commercially available radish (Raphanus sativus, Early scarlet globe) and mustard (Brassica juncea Florida broadleaf), at a 50:50 ratio, were used for the routine propagation of GPA (Myzus persicae) at 22°C in a growth chamber programmed for 14-h-light (100 µE m^–2 s^–1) and 10-h-dark cycle. All experiments reported in this article were performed at least three times with similar results, unless noted otherwise.

Arabidopsis Mutants

The pad3-1 (Zhou et al., 1999), pad4-1 (Glazebrook et al., 1997), cpr5 (Bowling et al., 1997), snc1 (Zhang et al., 2003), npr1-1 (Cao et al., 1994), and sid2-2 (Wildermuth et al., 2001) mutants used in this study are in the ecotype Columbia background. The ssi2, ssi2 nahG, and nahG plants are in the ecotype Nössen background (Shah et al., 1999, 2001). The pad4 T-DNA insertion line (SALK_089936) that was identified from the Salk collection is in the ecotype Columbia background (http://signal.salk.edu/).

No-Choice Test

A no-choice test was used to assay aphid growth on wild-type and mutant plants. Approximately 4-week-old Arabidopsis plants were used in the bioassay with a clonally propagated GPA population. For the no-choice test each Arabidopsis plant received 15 mature apterous aphids at the center of the rosette and the plants were incubated at 22°C as described above. Two days later, the plants were harvested and number of aphids residing on each plant was counted. Student's t tests were performed using SigmaPlot version 5.0 (SPSS).

Histochemistry and Microscopy

Leaf samples for trypan blue staining were processed and analyzed as described previously (Rate et al., 1999).

Chlorophyll Extraction and Estimation

Leaves were ground in a mortar with a pestle in the presence of liquid nitrogen. Chlorophyll was extracted with an extraction buffer consisting of an 85:15 (v/v) mix of acetone:Tris-HCl (1 M; pH 8.0 in water). The absorbance of the extract was recorded at 664 and 647 nm against an extraction buffer control and the chlorophyll content calculated as described previously (Lichtenthaler, 1987).

DNA and RNA Analysis

DNA for the PCR analysis was extracted from leaves as described previously (Konieczny and Ausubel, 1993). A transgenic Arabidopsis line (Salk_089936), which contains a T-DNA insertion within the PAD4 gene, was identified in the Salk collection (http://signal.salk.edu/). Multiplex-PCR analysis was performed on the segregating plant material to identify plants homozygous for the T-DNA insertion. The PAD4-F (5'-GCTCTCCTCTGCTGGAAACC-3'), PAD4-R (5'-TTTTCTCGCCTCATCCAACCA-3'), and T-DNA left border primer (5'-GCGTGGACCGCTTGCTGCAAC-3') were used for the multiplex PCR. PCR was performed with the following conditions: 95°C for 5 min followed by 30 cycles of 95°C for 0.5 min, 65°C for 0.5 min, and 72°C for 2 min, with final extension at 72°C for 5 min. The PCR products were resolved on 1.2% agarose gel, stained with ethidium bromide, and visualized with a BioDoc-It system (UVP).

For RNA extraction, leaf material from uninfested and GPA-infested plants was harvested and quick-frozen in liquid nitrogen. RNA was extracted by a guanidine-phenol method (Chomczynski and Sachhi, 1987). The isolated RNA was purified using the RNeasy Mini kit (Qiagen), spectrophotometrically quantified at 260 nm and subsequently used in the reverse transcription (RT)-PCR reactions. RT-PCR analysis was performed with the Superscript One-Step RT-PCR kit (Invitrogen). The RT reaction was carried out at 50°C for 30 min in a 20-µL reaction with 100 ng of the total RNA as template as recommended by the manufacturer. PCR conditions for the ACT8 (At1g49240), SAG12 (At5g45890), SAG13 (At2g29350), SAG21 (At4g02380), SAG27 (At2g44300), and PAD4 (At3g52430) were as follows: 95°C for 5 min followed by 25 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 1 min with a final extension at 72°C for 5 min. The ACT8-F (5'-ATGAAGATTAAGGTCGTGGCA-3') and ACT8-R (5'-TCCGAGTTTGAAGAGGCTAC-3'), SAG12-F (5'-TCTCGTCCACTCGACAATGAA-3') and SAG12-R (5'-AGCTTTCATGGCAAGACCACA-3'), SAG13-F (5'-CAAGATGGAGTCTTGGAGGCA-3') and SAG13-R (5'-GGAAAAACCGTTAACAGTGGA-3'), SAG21-F (5'-CCAATGCTATCTTCCGACGTG-3') and SAG21-R (5'-GAACCGGTTTCGGGTCTGTAA-3'), SAG27-F (5'-TCCTGGCCCTGAAGTAGAAA-3') and SAG27-R (5'-GTCCCGCAAGAACCTGTCC-3'), and PAD4-F (5'-GCTCTCCTCTGCTCGGAAACC-3') and PAD4-R (5'-TTTTCTCGCCTCATCCAACCA-3') gene-specific primers were used for PCR amplification of ACT8, SAG12, SAG13, SAG21, SAG27, and PAD4, respectively.

	ACKNOWLEDGMENTS

 
We thank Sharon McClung for providing us with the GPA colony; Drs. Fred Ausubel and Xin Li for providing the sid2 and snc1 mutants; the Arabidopsis Biological Resource Center at Ohio State University for providing the pad4 mutants; and Drs. Jane Parker, Sonny Ramaswamy, Ruth Welti, and Judith Roe, and members of the Shah lab, for critically reading this manuscript.

Received August 24, 2005; returned for revision October 7, 2005; accepted October 12, 2005.

	FOOTNOTES

 
¹ This work was supported by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture (agreement no. 2004–35301–14506); the National Science Foundation (grant no. MCB0416839); a Kansas State University Plant Biotechnology Center seed grant; the Kansas Idea Networks of Biomedical Research Excellence Grant (fellowship nos. K–INBRE and RR16475 to C.K.); and the Terry Johnson Cancer Center (fellowship to C.K.). This is Kansas Agricultural Experimental Station contribution 05–333–J.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jyoti Shah (shah{at}ksu.edu).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070433.

^* Corresponding author; e-mail shah{at}ksu.edu; fax 785–532–6653.

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