Free Access A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana Pablo Tornero, Department of Biology andSearch for more papers by this authorJeffery L. Dangl, Corresponding Author Department of Biology and Curriculum in Genetics, Coker Hall 108, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA For correspondence (fax: +1 919 962 1625; email: dangl@email.unc.edu)Search for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Measuring the growth of pathogenic bacteria in leaves is a mainstay of plant pathology studies. We have made significant improvements to standard methods that will not only increase the throughput but also reduce the space limitations. Additionally, the method described here is as accurate as the standard method. Briefly, we infected leaves by dipping whole seedlings of Arabidopsis into a bacterial solution containing a surfactant. After harvest, the seedlings were then simply shaken in buffer. The resulting bacterial solutions were diluted in microtitre plates and spotted onto agar plates. Colony-forming units were then counted 40 h after plating. Therefore, we have eliminated most of the labour-intensive steps involved in measuring the growth of bacteria in Arabidopsis, and describe a method that could be automated. The assay is sensitive enough to detect small differences between pathogens or ecotypes. Introduction The study of the interactions between plants and their pathogens is a strong and vibrant field in modern biology. Measuring the growth of pathogens in planta is critical to evaluate the response of the plant to a known quantity of pathogen. We focus on the interaction between Arabidopsis thaliana (Arabidopsis) and strains of Pseudomonas syringae (Ps, Glazebrook etal., 1997). Four cloned resistance genes (R) condition resistance to bacterial strains expressing corresponding, cloned avirulence (avr) genes from four strains of P. syringae have been characterized (reviewed by Jones, 2001; Nimchuck etal., 2001). Additionally, there are a number of virulent Ps strains that do not express these genes and cause disease on Arabidopsis. Thus, this interaction also provides an excellent model for dissecting the virulence mechanisms of P. syringae. Landmark efforts in the early 1990s (Debener etal., 1991; Dong etal., 1991; Whalen etal., 1991) described the interactions between these two organisms. Specifically, Whalen etal. (1991) described different procedures to inoculate Arabidopsis, namely pressure infiltration, dip, vacuum infiltration, and spray. Pressure infiltration continues to be the most commonly used method for measuring growth of bacteria in planta. In this method, leaves of adult, 4–5-week-old, short-day plants are inoculated. After that, a measured fragment of the leaf is detached with a cork borer, ground, and the bacteria serially diluted and plated in a agar plate on the appropriate selective medium (e.g. Morel and Dangl, 1998). For analyses of large numbers of plant–bacterial strain combinations, the amount of labour quickly becomes overwhelming. We revisited the dip inoculation protocol previously described (Whalen etal., 1991; Wanner etal., 1993), in order to facilitate our analyses of bacterial growth in a large number of mutant and transgenic lines of Arabidopsis. We optimized this method using small plants (2-week-old, 8 h day) to both increase the number of data points generated per unit of growth space and reduce the amount of labour. We have also developed a new simple method for measuring bacterial growth following dip inoculation of plants. We believe that these modifications will improve considerably the time and space needed to quantify growth of pathogenic bacteria in Arabidopsis. Seeds are sown in a pot, covered with nylon mesh, and grown under 8 h day conditions (Figure 1). Inoculation is performed 14 days after transfer to the growth chamber. Several factors were tested and found to be critical for reproducibility. First, the inoculum is prepared with bacteria grown on plates with the appropriate antibiotic for 24 h. Bacteria cultivated in liquid media do not generate reproducible results (data not shown). Second, once the bacteria have been resuspended in 10 mm MgCl2, Silwet L-77 is added at a final concentration of 200 µl l−1. This surfactant was also found to enhance the transformation of Arabidopsis by Agrobacterium tumefaciens (Clough and Bent, 1998). Third, because P. syringae can infect the leaf via stomata (Goto, 1992), we kept a transparent lid on the tray of pots after inoculation because stomatal opening is dependent on high humidity. We found that the uniformity of the inoculation was improved if we left the lid closed for 1 h. When high humidity was maintained for longer periods (24 h), the resistance response was reduced (data not shown). With these modifications, P. syringae is able to reproducibly infect Arabidopsis by dipping. Macroscopic symptoms in Arabidopsis thaliana 5 days post-inoculation (dpi) with Pst (avrRpm1) by dipping. The left part of the pot contains wild-type Col-0 plants, and the right part the mutant rpm1-1. Note that rpm1-1 is glabrous (Oppenheimer etal., 1991). We used P. syringae pathovar tomato, isolate DC3000 (Pst), which is an aggressive pathogen on Arabidopsis. We optimized bacterial concentration by using the concentration that did not produce a visual response during an incompatible (plant resistant) interaction and also yielded the highest difference in growth of bacteria between compatible (plant susceptible) and incompatible interactions. In our case, this concentration is OD600 = 0.05 (approximately 2.5 × 107 colony-forming units per ml (cfu ml−1)). This concentration of bacteria is higher than the inoculum generated in nature when rain runs through an infected leaf (105−107 cfu ml−1; Goto, 1992). However, the traditional pressure infiltration method typically uses a starting concentration of approximately 105 cfu ml−1 for growth determinations, and this produces inoculum levels of 103−104 cfu cm−2 of leaf (e.g. Morel and Dangl, 1998). This inoculum corresponds to roughly 2.5 × 102−2.5 × 103 cfu mg−1 of leaf (1 cm2 of a 3-week-old leaf weighs approximately 4 mg, data not shown). This number is very close to the effective inoculum of 103−104 cfu mg−1 we use here. Figure 1 shows symptoms 5 days post-infection (dpi) for plants that have been dip-inoculated using the method described above with Pst (avrRpm1) (Dangl etal., 1992). The plants growing in the left half of the pot are plants of Arabidopsis accession Col-0, which express RPM1 (Grant etal., 1995) and are thus resistant to this particular strain of Pst. At this bacterial concentration, there are no visible lesions in Col-0 and growth of the bacteria is suppressed. We used trypan blue staining to aid in the visualization of the hypersensitive response (HR) in plants. The HR is a programmed cell death process associated with some resistance response (reviewed in Morel and Dangl, 1997). Uninoculated leaves of Col-0 do not stain positively for trypan blue, except along the veins (data not shown). In contrast, leaves of Col-0 inoculated with Pst (avrRpm1) show occasional HR foci that stain positively for trypan blue as early as 8 h post-inoculation (hpi). Plants carrying the rpm1-1 mutant allele (Grant etal., 1995) are shown in Figure 1, growing in the right half of the pot. In the absence of active RPM1, Pst (avrRpm1) is able to thrive. The disease symptoms are apparent (Figure 1) within 5 dpi. There is no cell death in rpm1-1 as determined by trypan blue staining at 8 hpi. After 1 day, micro-colonies of bacteria were visible under epi-fluorescence, but still no cell death was observed. Therefore, the expected responses (determined by pressure infiltration) for different lines of Arabidopsis were observed in response to dip inoculation of Pst (avrRpm1). We routinely used this method to evaluate different Arabidopsis mutants that show qualitative differences in resistance against Pst (avrRpm1). We were able to successfully discriminate between several levels of resistance, such as those exhibited by strong and weak loss of function alleles of RPM1 (Tornero and Dangl, unpublished results). Nevertheless, once we had found qualitative differences with this method, we found ourselves switching to hand inoculation of older plants in order to generate quantitative data. As the standard method requires 4–5-week-old plants, this represented a loss of time and resources. In our system, the plants are too small to punch leaf discs of known size, so we decided to express the number of bacteria per fresh weight of the plant. Three seedlings per replicate were removed from pots containing inoculated plants. Care was taken to avoid taking soil or roots. These seedlings were placed in pre-weighed 1.5 ml microfuge tubes containing 200 µl of MgCl2. The tubes were weighed again to determine the amount of plant tissue for each sample. Data were collected in quadruplicate. Thus, each data point is the mean and standard error of four independent measurements and required 12 seedlings. The tubes were then processed with a grinder (Dual Range Stirrer with a custom-made stainless steel tip, Caframo Ltd, Ontario, Canada) as described previously (e.g. Morel and Dangl, 1998). With these changes in the method, we are able to use less space in the short-day chambers (a limitation in most laboratories) and less time. Thus, in our method we use a tray (28 × 54 cm) holding up to 20 pots (8.9 cm diameter) for 3 weeks. This allows the analysis of 20 plant lines with one pathogen. With pressure infiltration, we used the same surface for 5 weeks growing 36 plants (9 × 4 plant matrix), which resulted in data for nine plant lines (using four individuals per analysis). The improvement is considerable, from 1.8 lines per week/unit surface in the hand inoculation, to 6.7 lines per week/unit surface in the dip inoculation. There is also an important saving in hands-on time. Thus, hand inoculation of the same nine plant lines takes between 20 and 30 min, while dip inoculation of the same growing surface (20 pots) takes between 5 and 10 min. Therefore, the time required for hand inoculation is 2.2–3.3 min line−1 versus 0.25–0.5 min line−1 for dip inoculation, a significant improvement. However, the most important benefit is obtained in time required for sample handling. As the samples are shaken for 1 h, this time is used for preparation of the dilutions in the next step. With 20 lines, our estimation of the time needed for grinding the tissue is 3 h of hands-on work. We took 1 h after inoculation as time zero, as this is the time when we open the lid. The intention of this measurement is to assess the uniformity of the inoculation. In our case, the differences in phenotypes between control incompatible and compatible interactions are obvious at 3 and 5 dpi (Figure 1). However, we prefer not to measure beyond day 3, as by 4 dpi, some of the leaves in the compatible interactions are completely desiccated but still carry viable bacteria. This is a source of error when plants are picked, as we express growth per weight, so we decided to avoid it. If an extended time course is needed, a low initial inoculum (as low as OD600 = 5 × 104 cfu mL−1) is recommended. We also designed a method to rapidly extract bacterial cells from infected leaves, further reducing the amount of labour required to process samples. The same number of seedlings per sample as above was used. In order to improve the reproducibility, we increased the volume of the buffer to 1 ml per tube, and added 0.2% v/v Silwet L-77. Up to 96 microfuge tubes were then placed into a single 2 l flask and shaken for 1 h at 28°C, 18.7 g (250 rpm). Shaking for longer periods of time (3 h) did not result in an observable increase in the number of bacterial cells recovered from leaves of Arabidopsis (data not shown). Washing leaves to remove any bacteria possibly growing on the surfaces prior to shaking also did not affect the number of cells recovered. After bacterial extraction, we used the dilution procedure already established to measure the number of cfu per ml following pressure infiltration. Thus, we prepared a microtitre plate by adding 180 µl of 10 mm MgCl2 to each well. Aliquots (20 µl) of bacterial solution were removed from each tube and independently added (undiluted from the tube) to the wells in the first column of the microtitre plate. We then used a multi-channel pipette to make serial 10-fold dilutions in wells corresponding to columns 2–6. Drops (2 µl) from each well were spotted onto a single 150 mm agar plate carrying the appropriate antibiotics. Drop sizes larger than 2 µl often mixed with one another. Thus, one agar plate is sufficient for measuring 96 dilutions of bacteria. The number of colony-forming units is then counted as described below. We have also used an alternative method to spotting 2 µl drops onto plates. A 96-pronged stamp can also be used to spot a small aliquot of each bacterial dilution onto the plates in a single motion. The weight of the stamp is enough to make a reproducible impression in the agar without breaking the surface. Between different microtitre plates, the stamp is briefly dipped in ethanol and then blotted three times onto absorbent paper. No cross-contamination results using this treatment (data not shown). The agar plates are then incubated at 25°C for 40 h and the number of colony-forming units is counted. We can reliably count dilutions that have between 1 and 20 per drop. The total number of bacteria (cfu/mg fresh weight) is calculated by multiply the number of colonies by a constant ‘k’ (see Experimental procedures) adjusting the dilution, and dividing by the weight of the tissue as described in Experimental procedures. Figure 2 indicates that recovery of bacterial cells at 3 dpi by shaking is as effective as that obtained by grinding leaf tissue. The data presented on the left show the growth of Pst (avrRpm1) on Col-0, and the data on the right show growth of the same bacteria on rpm1-1. For the day 0 data (white columns), the number of bacterial cells was determined in each genotype of plant at 1 hpi by the method described here. These results indicate that the inoculations were uniform between genotypes of plants. The remaining columns indicate that, by 3 dpi, Pst (avrRpm1) can successfully grow in rpm1-1 plants, but not in Col-0. The decrease in the number of colony-forming units at 3 dpi, relative to 0 dpi, in Col-0 is most likely due to the increase in leaf mass in the ratio cfu mg−1. More importantly, the data clearly show that the recovery of bacterial cells from rpm1-1 plants by shaking (column 2) or grinding (column 3) is comparable. Column 4 represents the bacteria released from rpm1-1 plants that were first shaken for 1 h, the tissue briefly blotted, and then ground using a grinder. Although it appears that a significant number of bacterial cells remain in the plant, this represents only approximately 10% of the total number of bacterial cells recovered by shaking alone. Therefore, shaking of plants to recover bacterial cells from plants is as efficient as grinding the leaf tissue, but requires significantly less labour. Comparison of different methods of extracting bacteria from Arabidopsis. Plants of Col-0 (left group of columns) or rpm1-1 (right group of columns) were inoculated by dipping with Pst (avrRpm1). At day 0 (white columns) and day 3 (light grey columns), bacteria were extracted from the plants by shaking. After the buffer had been removed, the tissue was blotted and ground with a grinder in order to quantify the remaining bacteria (black columns). Independent samples with no previous treatment were also ground (dark grey columns).Figure 3 shows that the method presented here is sensitive enough to measure small differences in the growth of different strains of Pst on Arabidopsis. Pst (avrRpm1) and Pst (avrRpt2) (Innes etal., 1993) grow to different levels on susceptible near-isogenic lines of Arabidopsis that carry mutations in the respective R genes, RPM1 and RPS2 (Bent etal., 1994; Grant etal., 1995; Mindrinos etal., 1994). In previous studies, growth of the bacterial strains was measured following pressure infiltration. We used the method described here to determine whether we could observe the same patterns of growth on these Arabidopsis lines. As seen in Figure 3, we detect the same differences in growth of the different strains of Pst on the different lines of Arabidopsis, i.e. Col-0 plants exhibit a more effective resistance to Pst (avrRpm1) than to Pst (avrRpt2). We realize that our results are somehow different to the ones reported previously. Thus, Ritter and Dangl (1996) reported a difference in growth of Pst (avrRpm1) in Col-0 of more than 10-fold between days 0 and 3. We speculate that dip inoculation, which reflects natural infection more accurately, is more sensitive to small differences between pathogens or plants. This idea was first proposed by Wanner etal. (1993). Also, Mittal and Davis (1995) reported no difference in growth of bacteria with and without the phytotoxin Coronatine in Arabidopsis when the bacteria were hand-infiltrated. However, the authors reported strong differences when the same bacteria were dip-inoculated. In our hands, the symptoms observed with the dip method are more pronounced that in the hand inoculation method. For instance, with Pst (avrRpm1) no macroscopic symptoms were observed in Col-0 at day 5 by dip inoculation (Figure 1), as opposed to hand inoculation where mild disease symptoms are frequent (data not shown). Conversely, the symptoms observed in the rpm1 mutant inoculated with Pst (avrRpm1) (Figure 1) are more severe than the equivalent hand inoculation (data not shown). Comparison of different pathogens in the same plants. Plants of Col-0, rpm1-1 or rps2-101c were inoculated by dipping with Pst, Pst (avrRpm1) or Pst (avrRpt2). At day 0 (white columns) and day 3 (light grey columns), bacteria were extracted from the plants by shaking. An independent repetition was performed. It is important to mention several aspects of the repetitions. Data from independent experiments should not be pooled together. While genotype-dependent growth differences are maintained, a Student\'s t test (with α = 0.05) recognized parallel data from independent experiments as statistically different in 3 out of 10 pair wise comparisons. This phenomenon was observed in several systems, including data from tissue culture experiments and hand inoculations of bacteria in Arabidopsis (data not shown). Incidentally, the same t test does not recognize differences in bacterial levels at day 0 within an independent experiment. Conversely, differences between days 0 and 3 in each case are catalogued as significant, with the exception of the incompatible interaction between Pst (avrRpm1) and Col-0 in both experiments, and the interaction between Pst (avrRpt2) and Col-0 in the second repetition. Most importantly, the differences between Col-0 and the respective r gene mutants were statistically significant when challenged with the same strain of bacteria. We also used the dip inoculation method to recapitulate results observed in the interactions between Pst (avrB) and RPM1 (Grant etal., 1995; Tamaki etal., 1991) Pst (avrPphB) and RPS5 (Mansfield etal., 1994; Warren etal., 1998) and Pst (avrRps4) and RPS4 (Gassmann and Staskawicz, 1999; Hinsch and Staskawicz, 1996) (data not shown), as well as the interactions between different Pst isolates and mutant lines lsd1, pad4, eds1, nim1, nahG and some double mutant combinations (Rusterucci etal., 2001; Aviv etal., 2001). Our method can also be used to detect small differences in growth of bacteria that cannot be detected using the pressure infiltration method. No major differences in growth of Pst (avrRpm1) on different RPM1-expressing ecotypes of Arabidopsis have been reported. However, using the dip inoculation method, we observed a several fold higher growth of Pst (avrRpm1) in plants of Landsberga erecta relative to Col-0 (Figure 4; first four columns). Our data suggest that the cotyledons of Landsberga erecta were considerably more susceptible than the rest of the plant (Figure 4; columns 5 and 6). This suggests a novel developmental control of RPM1 function in La-er. Note that the number of cotyledons per mg is higher than the number of true leaves per mg, explaining the observation that the bacterial titre in cotyledons is higher than in the total plant. We used trypan blue staining of infected cotyledons of Landsberga erecta at 5 dpi and observed the presence of microcolonies. No microcolonies were observed in infected leaves of Col-0. Comparison of different ecotypes infected with the same pathogen. Plants of Col-0 and Landsberga erecta (La-er) were inoculated by dipping with Pst (avrRpm1) and the bacteria extracted by shaking. At day 3, plants of La-er were either processed as whole seedlings (La-er vs Pst (avrRpm1)) or each plant was divided into cotyledons (La-er vs Pst (avrRpm1), cotyledons) and the rest of the plant (La-er vs Pst (avrRpm1), rest). Note that the day 0 data for La-er are from the whole seedling in all cases. To summarize, this method is fast, reliable, and can unveil small differences between pathogens and ecotypes of Arabidopsis. There are disadvantages of this method, mainly the fact that the plant cannot be recovered, as opposed to the detachment and grinding of a single leaf. This aspect may be limiting on occasions were few plants are available. However, we believe that the enormous saving of space, time of growth until experimentation, hands-on time, and the reduction in the fatigue of the researcher overcomes these problems. The mutants rpm1-1 (Grant etal., 1995) and rps2-101c (Yu etal., 1993) have been described previously. Note that rpm1-1 was isolated in a glabrous background (Oppenheimer etal., 1991) as an independent marker. The plants are sown in pots (8.9 cm diameter) covered with a mesh (bridal veil, purchased in a local fabric store, with the threads separated 1 mm), and the mesh is secured with a rubber band. Plants are grown under a short-day regimen, as described previously (Ritter and Dangl, 1996). A small number of plants are sown, in order to avoid crowding. In practice, we thin the plants at day 10 (after transfer to the growth conditions) until 50–100 plants are left. The inoculation is done at day 14. Pseudomonas syringae pv. tomato DC3000 (Pst) derivatives containing pVSP61 (empty vector, no avr gene) or avrRpm1 or avrRpt2 (in the vector pVSP61) were used and maintained as described previously (Ritter and Dangl, 1996). Approximately 24 h prior to inoculation, a generous sample of the bacteria growing on a plate was plated onto a fresh plate of KB medium (King etal., 1954) with the appropriate antibiotics. The inoculum was then distributed using a spreader in order to obtain a lawn of bacteria. Once the bacteria have grown for 24 h at 25°C, 10 ml of 10 mm MgCl2 is added to the plate. Ten minutes later, the bacterial suspension is washed out of the plates with a pipette, the OD600 is measured, samples adjusted to OD600= 0.05, and Silwet L-77 (OSi Specialties Inc., Danbury, Connecticut, USA) is added to a final concentration of 200 µl l−1. The pot is then turned upside-down and submerged in the bacterial solution for 10 sec. In order to ensure that all the plants are dipped, the pot is submerged approximately 3 cm above the soil. We prepare aliquots of 1 l per 20 pots (8.9 cm of diameter each), discarding the remaining. We inoculate plants at the beginning of the short day to ensure reproducibility. Once the inoculation has been completed, the plants are moved to the short-day chamber with a transparent lid covering the plants. One hour after the inoculation, the lid is removed and the samples for day zero are taken. Briefly, three whole seedlings, without roots, are placed into a pre-weighed 1.5 ml tube containing 10 mm MgCl2 and 0.2% v/v Silwet L-77 and the weight is recorded. Four tubes are prepared for each data point. The tubes are then introduced in a 2 l flask, and this flask is shaken at 28°C, 18.7 g (250 rpm), for 1 h. After this time, 20 µl from each tube are added to the wells of a microtitre plate containing 180 µl of 10 mm MgCl2, and serial 10-fold dilutions are prepared with a multi-channel pipette. The bacteria are spotted onto a 150 mm Petri plate of KB containing the appropriate antibiotics with the help of a 96-pronged stamp, and the plate is incubated at 25°C. Forty hours later, the number of colonies are counted. We count the dilution that gives us between 1 and 20 colonies. The number of cfu mg−1 fresh weight is determined by the formula: cfu mg − 1 FW  =  k (N  ×  10d  −  1)/(weight of the tissue) where N is the number of colonies counted in the dilution number d, and k is a constant, calculated as follows. One colony in the first dilution would indicate that the concentration in the first well was 1 cfu/0.6 µl, as this is the volume of liquid delivered by the stamp (determined empirically, data not shown). As this is from a 10-fold dilution, it is equivalent to 10 cfu/0.6 µl from the original tube. Assuming that the volume of the plants is negligible in comparison to the 1000 µl of buffer, this equals 16 667 cfu per tube. Therefore, the constant k is 16 667. Each data point is represented as the mean and standard error of the decimal logarithm of four repeats. Independent experiments were performed on different days to ensure that the method is robust. We thank Susanne Kjemtrup and Daniel Aviv for their input into the development of this method, and Daniel Aviv, Jeff Chang and David Hubert for comments on the manuscript. This work was funded by National Science Foundation grant IBN-9724075 to J.L.D. and a Postdoctoral Fellowship from the Spanish Ministry of Science and Education to P.T.Inoculation by dipping.Inoculate 2-week-old plants, growing in small pots covered with amesh. Grow the bacteria in plates, not in liquid. Use 1l per20 pots, and then discard the remainder. Be sure that the pots arewell watered the day before the inoculation.MethodDay 11 Grow inoculum in plates. Be sure that there is enough to producea lawn of bacteria.Day02 Add 10ml of 10mM MgCl2 per plate and washwith the pipette. If the buffer is added allowed to sit for10min, it will be easier.3 Recover the bacteria with a pipette and add to a 250mlflask containing 200ml of 10mM MgCl2, dilute1/10 for measure.4 Measure the OD600, adjust to 0.05. Add Silwet L-77,200mll 1.5 Dip pots in the solution for about 10sec. Be sure that allthe plants are submerged.6 Cover the plants and put them into the growth chamber.7 Remove the lid after 1h. Appendix2Growth curves (dipping).Method1 After dipping, keep the lid closed for 1h (time 0 is1h post-inoculation).2 Prepare tubes (four repeats per data point) with 1ml10mM MgCl2+0.02% Silwet L-77.3 Weigh the tubes (±1mg) and recordweight.4 Pick three plants per repeat (whole aerial part, avoid soil) andreturn the plants to the chamber.5 Weigh the tubes (±1mg) and recordweight.# 6 Put the tubes in a 2l flask in the 28°C shaker,250revmin 1, 1h.7 Bring the Petri plates (large plates) to room temperature.8 Prepare the microtitre plates, adding 180µl of10mM MgCl2, six wells per repeat.9 When the hour is up, add 20µl of the supernatant towell1.10 Once all wells1 are complete, transfer 20µlfrom well1 to well2, using a multi-channel pipette. Mixbefore and after pipetting and always change tips.11 Repeat from wells 2 to wells 3, until wells 6.12 Label the Petri plates.13 Prepare a container with EtOH (empty yellow box) and two papertowels.14 Clean the 96-pin stamps with EtOH, blot on one towel, and thenon another.15 Dip the stamp in the microtitre plate and then blot on the Petriplate. The weight of the stamp is enough to make an impressionwithout breaking the agar.16 Clean as before.17 Incubate for 48h at 25°C.18 Count. The first dilution is multiplied by 50000/3 anddivided by fresh weight in mg, resulting incfumg 1 fresh weight. FigureS1.Trypan blue stainingof leaves showing microscopic symptoms. FigureS2.Scan of an agarplate showing the colonies. FigureS3.Microscopy ofcotyledons stained with trypan blue. FigureS4a.Raw data, includingrepeats and statistical test. FigureS4b.Raw data, includingrepeats and statistical test. FigureS4c.Raw data, includingrepeats and statistical test. FigureS4d.Raw data, includingrepeats and statistical test. FigureS4e.Raw data, includingrepeats and statistical test. FigureS4f.Raw data, includingrepeats and statistical test. FigureS4g.Raw data, includingrepeats and statistical test. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.Parker, J.E., Dangl, J.L. (2001) Runaway cell death, but not Basal Disease resistance, in Isd1 is SA- and NIM/NPR1-Dependent. Plant J., in press.Leung, J., Staskawicz, B.J. (1994) RPS2 of Arabidopsis thaliana. A leucine-rich repeat class of plant disease resistance genes. Science, 265, 1856– 1860. Clough, S.J. & Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735– 743. Wiley Online LibraryTaylor, J.D., Vivian, A. (1992) Functional homologs of the Arabidopsis RPM1 disease resistance gene in bean and pea. Plant Cell, 4, 1359– 1369.Arnold, M., Dangl, J.L. (1991) Identification and molecular mapping of a single Arabidopsis thaliana locus determining resistance to a phytopathogenic Pseudomonas syringae isolate. Plant J. 1, 289– 302.Davis, K.R., Ausubel, F.M. (1991) Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene. Plant Cell, 3, 61– 72.Rogers, E.E., Ausubel, F.M. (1997) Use of Arabidopsis for genetic dissection of plant defense responses. Annu. Rev. Genet. 31, 547– 569. Goto, M. (1992) Fundamentals of Bacterial Plant Pathology. New York: Academic Press Inc.Innes, R.W., Dangl, J.L. (1995) Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science, 269, 843– 846. Hinsch, M. & Staskawicz, B.J. (1996) Identification of a new Arabidopsis disease resistance locus RPS4, and cloning of the corresponding avirulence gene, avrRps4, from Pseudomonas syringae pv. pisi. Mol. Plant–Microbe Interact. 9, 55– 61.Bisgrove, S.R., Staskawicz, B.J. (1993) Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. J. Bacteriol. 175, 4859– 4869. Jones, J.D.G. (2001) Putting knowledge of plant disease resistance genes to work. Curr. Opin. Plant Biol. 4, 281– 287. Ward, M.K., Raney, D.E. (1954) Two simple media for the demonstration of phycocynin and fluorescein. J. Lab. Clin. Med. 44, 301– 307.Bennett, M.A., Stewart, R. (1994) Characterization of avrPphE, a gene for cultivar-specific avirulence from Pseudomonas syringae pv. phaseolicola which is physically linked to hrpY, a new hrp gene identified in the halo-blight bacterium. Mol Plant–Microbe Interact. 7, 726– 739.Yu, G.-L., Ausubel, F.M. (1994) The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell, 78, 1089– 1099. Mittal, S. & Davis, K.R. (1995) Role of the phytotoxin Coronatine in the infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. Mol Plant–Microbe Interact. 8, 165– 171. Morel, J.-B. & Dangl, J.L. (1997) The hypersensitive response and the induction of cell death in plants. Cell Death Differ. 19, 17– 24. Morel, J.-B. & Dangl, J.L. (1998) Suppressors of the Arabidopsis lsd5 cell death mutation identify genes involved in regulating disease resistance responses. Genetics, 151, 305– 319.Chang, J.H., Dangl, J.L. (2001) Knowing the dancer from the dance: R gene products and their interactions with other proteins from host and pathogen. Curr. Opin. Plant Biol. 4(4), 288– 294 Esch, J., Marks, M.D. (1991) A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell, 67, 483– 493. Ritter, C. & Dangl, J.L. (1996) Interference between two specific pathogen recognition events mediated by distinct plant disease resistance genes. Plant Cell, 8, 251– 257.Dangl, J.L., Parker, J.E. (2001) The disease resistance signaling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in Arabidopsis. The Plant Cell, 13, 2211– 2224.Kobayashi, D.Y., Keen, N.T. (1991) Sequence domains required for the activity of avirulence genes avrB and avrC from Pseudomonas syringae pv. glycinea. J. Bacteriol. 173, 301– 307.Mittal, S., Davis, K.R. (1993) Recognition of the avirulence gene avrB from Pseudomonas syringae pv. glycinea by Arabidopsis thaliana. Mol. Plant–Microbe Interact. 5, 582– 591.Holub, E., Innes, R.W. (1998) A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell, 10, 1439– 1452.Bent, A.F., Staskawicz, B.J. (1991) Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell, 3, 49– 59.Katagiri, F., Ausubel, F.M. (1993) Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene avrRpt2. Mol. Plant–Microbe Interact. 6, 434– 443. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Please check your email for instructions on resetting your password. 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