Abstract
We present evidence that susceptible Arabidopsis plants accelerate their reproductive development and alter their shoot architecture in response to three different pathogen species. We infected 2-week-old Arabidopsis seedlings with two bacterial pathogens, Pseudomonas syringae and Xanthomonas campestris, and an oomycete, Peronospora parasitica. Infection with each of the three pathogens reduced time to flowering and the number of aerial branches on the primary inflorescence. In the absence of competition, P. syringae and P. parasitica infection also increased basal branch development. Flowering time and branch responses were affected by the amount of pathogen present. Large amounts of pathogen caused the most dramatic changes in the number of branches on the primary inflorescence, but small amounts of P. syringae caused the fastest flowering and the production of the most basal branches. RPS2 resistance prevented large changes in development when it prevented visible disease symptoms but not at high pathogen doses and when substantial visible hypersensitive response occurred. These experiments indicate that phylogenetically disparate pathogens cause similar changes in the development of susceptible Arabidopsis. We propose that these changes in flowering time and branch architecture constitute a general developmental response to pathogen infection that may affect tolerance of and/or resistance to disease.
One way that plants can respond to pathogen infection is through an induced resistance response called R gene resistance. When a plant has an R gene that confers resistance to an infecting pathogen, the plant initiates extensive biochemical and structural defense mechanisms, including the production of phytoalexins and pathogenesis-related proteins, the strengthening of cell walls, local cell death, and systemic acquired resistance (Dangl and Jones, 2001). Plants that are “susceptible,” i.e. do not have R gene resistance to a particular pathogen strain, exhibit many defenses similar to R gene resistance, but are slower in their expression of these responses (Yang et al., 1997; Maleck et al., 2000). Like R gene-resistant plants, susceptible plants have increased levels of salicylic acid (SA; O'Donnell et al., 2001), cell death, and defense mechanisms characteristic of systemic acquired resistance (Glazebrook, 2001). In addition, infected, susceptible plants can exhibit increased levels of jasmonic acid (JA), auxin, and ethylene (Dong, 1998; Lund et al., 1998; O'Donnell et al., 2003).
These responses of plants to pathogen infection bear some similarity to responses to abiotic stress. Both can involve cell death (Beers and McDowell, 2001), ethylene, SA, and JA (Wang et al., 2002) and cause changes in the expression of some of the same transcription factors (Chen et al., 2002). One way in which plants respond to abiotic stresses is to accelerate their transition to reproduction. Researchers have noted that plants flower faster in response to shade, overcrowding, low nutrients, drought, heat, and low light quality (Casal and Smith, 1989; Levy and Dean, 1998). Arabidopsis, in particular, has been shown to flower faster with low nutrients (Martinez-Zapater et al., 1994) and with shade (Halliday et al., 1994). Given the similarity between responses to pathogen infection and abiotic stress, it is possible that plants also accelerate their reproduction in response to pathogen infection, although whether they do so has not been explored.
There are several other reasons to expect that plants will respond to pathogen infection with changes in the timing of reproduction. First, one study reported that Arabidopsis infected with Pseudomonas syringae DC3000 had a higher probability of bolting under 8-h light conditions (Peters, 1999), suggesting that pathogen infection can alter the initiation of reproduction in plants. Second, life history evolution models predict that organisms faced with severe disease should evolve to reproduce more quickly (Minchella, 1985; Hochberg, 1992; Forbes, 1993; Agnew et al., 2000). In accordance with the predictions of life history models, some animals exhibit accelerated reproduction with parasite infection (Agnew et al., 2000). Finally, the hormones auxin, JA, and ethylene, which are elevated in infected plants, also have roles in plant development (Berleth and Sachs, 2001; Turner et al., 2002; Wang et al., 2002).
Changes in the timing of reproductive development in plants are often associated with changes in plant growth and branch architecture (Martinez-Zapater et al., 1995; van Tienderen et al., 1996; Bradley et al., 1997; Pineiro and Coupland, 1998; Pigliucci and Schmitt,1999). Changes in the timing of reproduction and in branch morphology can have important ecological and fitness consequences (Schoen and Dubuc, 1990; Diggle, 1995; Fishbein and Venable, 1996; Stowe et al., 2000; Schippers et al., 2001). For example, changes in developmental rates may affect age-related resistance, which is typically seen as an increase in the resistance of plants with development. One instance of this was reported by Kus et al. (2002), who found that in stressful conditions, where accelerated flowering occurred, young Arabidopsis plants exhibited early age-related resistance.
In this paper, we show that pathogen infection can alter time to flowering and branch architecture in susceptible Arabidopsis plants. We investigate the generality of these changes in development by examining the effects of three distantly related pathogens: P. syringae and Xanthomonas campestris, which are biotrophic bacterial pathogens, and Peronospora parasitica, which is a biotrophic oomycete. All of these pathogens occur in natural Arabidopsis populations (Tsuji and Somerville, 1992; Holub et al., 1994; Jakob et al., 2002). We also address how the amount of pathogen in a plant affects development and whether R gene resistance affects developmental responses to pathogen infection.
RESULTS
P. syringae Infection Alters Flowering Time and Branch Architecture in Susceptible Plants
To examine the effect of P. syringae on development, we infected susceptible plants (rps2) with P. syringae DC3000 + avrRpt2 at 2 weeks old. P. syringae infection significantly accelerated flowering time, decreased the number of aerial branches (branches above the rosette), increased the number of basal branches (branches from the rosette), and decreased the height of the primary inflorescence (Tables I and II). Because plant development can vary dramatically in different growth conditions, we evaluated the effects of infection both with and without competition. When grown with competitors, susceptible plants infected with P. syringae flowered on average 5.1 d faster than mock-treated plants and, without competition, flowered 2.5 d faster. Infected plants produced almost two fewer secondary aerial branches than mock-treated plants in the presence of competition and almost three fewer in the absence of competition. Infected plants also produced, on average, 1.1 more basal branches than mock-treated plants in the absence of competition. Another indication of increased development of the basal branches is the almost 2-fold increase in the number of siliques produced by the basal branches in infected plants compared with mock-treated plants.
Table I.
Trait means for susceptible (rps2) plants infected with P. syringae DC3000 + pLABL18 or given a mock treatment*
Trait
|
Competition
|
No Competition
|
||
---|---|---|---|---|
P. syringae | Mock | P. syringae | Mock | |
Flowering time (d) | 39.6***a | 44.6 | 37.8*** | 40.3 |
No. of aerial branches | 3.6*** | 5.5 | 3.6*** | 6.5 |
Height (cm) | 27.1*** | 35.1 | 41.7*** | 45.8 |
No. of basal branches | 0.3 | 0.1 | 6.2*** | 5.1 |
No. of siliques on the basal branches | 2 | 1 | 694*** | 362 |
a Asterisks indicate significant differences between pathogen-infected and mock-treated plants within competition treatments.
*, P < 0.05
***, P < 0.001.
Table II.
Effects of three pathogens on susceptible plant development**
Trait | P. syringae DC3000 | P. parasitica Noco2 | X. campestris 8004 |
---|---|---|---|
Accelerated flowering | ***a | * | *** |
Fewer aerial branches | *** | * | *** |
More basal branchesb | *** | n.s. | n.t.c |
More siliques produced by | *** | * | n.t. |
basal branchesb |
a Astericks indicate significant differences in means contrasts between infected and mock-treated susceptible plants in the indicated direction.
*, P < 0.05;
**, P < 0.01;
***, P < 0.001; n.s., not significant.
b Traits measured only in the absence of competitors in large pots. Plants rarely produced basal branches with competitors and in the small pots used in the X. campestris experiment.
c n.t., Not tested.
P. parasitica and X. campestris Infections Induce Similar Changes in Flowering Time and Branch Architecture in Susceptible Plants
To investigate whether the developmental changes we observed are specific to the bacterial pathogen P. syringae, we evaluated the effects of P. parasitica Noco2 and X. campestris pv campestris 8004 on susceptible plants. Both P. parasitica and X. campestris infections accelerated flowering and decreased the number of aerial branches (Tables II and III). In the absence of competition, P. parasitica infection also significantly increased the number of siliques produced by basal branches, indicating increased basal branch development. There was no significant difference in the number of basal branches produced with and without P. parasitica infection. Fewer basal branches were produced in the P. parasitica and X. campestris experiments compared with the P. syringae experiment, which likely reflects differences in the experimental conditions used for these different pathogens. Unlike P. syringae infection, P. parasitica and X. campestris infection did not significantly alter height.
Table III.
Trait means for susceptible (rpp5) plants infected with P. parasitica Noco2 or given a mock treatment and wild-type Columbia plants infected with X. campestris 8004 (OD600 = 0.03) or given a mock treatment**d
Trait
|
Competitiona
|
No Competition
|
X. campestrisb
|
Mock
|
||
---|---|---|---|---|---|---|
P. parasiticab | Mock | P. parasitica | Mock | |||
Flowering time (d) | 37.8*c | 39.1 | 35.8 | 36.4 | 46.1*** | 48.9 |
No. of aerial branches | 5.2 | 5.4 | 6.3* | 7.1 | 5.5*** | 6.9 |
Height (cm) | 32.3 | 32.7 | 40.3 | 41.2 | 28.8 | 28.0 |
No. of basal branches | 0.2 | 0.1 | 2.4 | 2 | 0.3 | 0 |
No. of siliques on the basal branches | 1.6 | 0.6 | 63* | 37 | n.d. | n.d. |
a Grown with six untreated RPP5-resistant plants.
b X. campestris and P. parasitica data are from a separate experiments with different experimental conditions.
c Asterisks indicate significant differences between pathogen-infected and mock-treated plants detected within competition treatment.
*, P < 0.05;
**, P < 0.01;
***, P < 0.001.
d n.d., Not determined due to the rarity of basal branches.
The Amount of P. syringae in Infected Leaves Affects Development
Next, we addressed whether the amount of pathogen affects the extent of developmental changes. To obtain variation in the amount of pathogen, we infected plants with four strains of P. syringae that vary in their growth in Columbia (Col). In addition to exploiting variation in the growth potential of strains, each strain was infected at three different initial doses, designated high, medium, and low.
Pathogen strain and initial dose did not affect symptoms or developmental responses independent of bacterial abundance 4 d after infection. That is, differences in the initial dose were overwhelmed by the different propensities of the strains to grow, and the identity of the strain had no effect over and above its growth. We tested for effects of initial dose and pathogen strain with analyses of covariance that included Strain, Dose, Strain × Dose, and, as a surrogate for the amount of pathogen 4 d after infection, the length of the second infected leaf as a covariate. Length of the second leaf 9 d after infection correlated strongly with the amount of pathogen across pathogen dose treatments (R = -0.96) and could be measured on the same plants as the developmental traits, unlike the amount of pathogen, which requires destructive sampling. There were no effects of Dose, Strain, or Dose × Strain for any of the traits measured. As a consequence, in further analyses, strain dose treatment means are treated as independent samples in our investigation of the relationship between pathogen abundance and plant traits.
The amount of P. syringae present in the infected leaves affected the percentage of infected leaf area that was visibly damaged (β = 0.90, P < 0.0001; Fig. 1A). Given the tight association between amount of pathogen and visible leaf damage, we cannot distinguish effects of disease symptoms from effects of the amount of pathogen present. As a consequence, we present results in terms of plant response to amount of pathogen, but the results could be interpreted equally well in terms of plant response to leaf damage.
Figure 1.
Effect of amount of pathogen present per infected leaf 4 d after infection. Black circles, Means for the compatible and non-host P. syringae strain dose combinations. Solid line, Regression line through these data. White circles, Three dose treatments for DC3000 + avrRpt2 to which the plants have R gene resistance. Rectangles, Three X. campestris dose treatments. The letters beside the markers indicate the strain (D, DC3000; E, ES4326; N, NPS3121; R, RM18.1; A, DC3000 + avrRpt2; X, X. campestris 8004) and dose (Low, Medium, and rHigh). Dashed line, Value of the trait for the mock treatment, which does not have a corresponding pathogen amount. Plants are wild-type Col. Each point is a mean of 19 replicates. A, Percentage of infected leaf area with disease symptoms 9 d after infection. B, Number of days to bolting. C, Number of secondary aerial meristems.
The amount of P. syringae present positively affected time to bolting (β = 0.86, P < 0.001) and time to flowering (β = 0.82, P = 0.001) but did not affect the number of rosette leaves on the day of bolting (P = 0.61). Given the pattern for bolting time and to better understand the effects of very low doses of P. syringae on the timing of development, we investigated whether the plants with very low doses of P. syringae bolted significantly faster than mock-treated plants (Fig. 1B). Based on a gap in the distribution of amount of P. syringae, we designated all treatments with less than 4.5 log colony-forming units of P. syringae per leaf as low. We determined whether plants in the group of low doses bolted faster than those in the mock treatment by calculating a one-way ANOVA with Treatment (strain dose combination), which included the 12 nonincompatible P. syrinage dose treatments and mock treatment. We then calculated a single means contrast between the mock treatment and the class containing low titers of P. syringae. Plants infected in the low group of P. syringae treatments bolted significantly faster than plants in the mock treatment (F = 5.02, degrees of freedom [df] = 1,234, P = 0.026).
The amount of P. syringae also affected branch architecture. The number of aerial meristems (Fig. 1C) and the number of aerial branches each decreased with greater amounts of pathogen (β = -0.94 and β = -0.86, respectively, P < 0.001 for both). Height was positively associated with the amount of P. syringae present (β = 0.59, P = 0.043). In addition, the point of origination on the primary branch of the first, uppermost aerial branch was significantly lower with increasing amounts of P. syringae (β = -0.9, P < 0.001).
Only a few plants produced basal branches in this experiment; as a consequence, no changes in the production of basal branches were evident. Because basal branching is likely to be affected by the amount of resources, we investigated the effect of initial dose of P. syringae DC3000 in plants grown in larger pots that permitted basal branching. Plants infected with a low dose of P. syringae produced more basal branches than plants infected with a high dose and also flowered more quickly (Fig. 2). There was no significant effect of dose on the number of aerial branches in this experiment.
Figure 2.
Mean numbers of days to flowering, aerial branches, and basal branches for rps2 plants grown in large pots and infected with a low, medium, or high dose of P. syrinage DC3000 + pLABL18 (OD600= 0.0001, 0.0003, and 0.001, respectively) or a mock treatment. Error bars = ses. Letters denote differences between treatments for P < 0.05.
The Amount of X. campestris Has a Different Effect on Arabidopsis Development
To explore the effect of amount of pathogen for a different kind of pathogen, in conjunction with the experiment employing the different P. syringae strains, we infected plants with three doses of a compatible X. campestris strain. The effect of X. campestris dose on plant development was similar to that of P. syringae for branch architecture but not for flowering time (Fig. 1). Larger amounts of X. campestris were associated with fewer aerial branches (means contrast between high and low doses, F = 11.2, df = 1,54, P = 0.0015), a lower point of origin on the primary branch for the uppermost aerial branch (F = 10.3, df = 1,54, P = 0.002), and taller primary inflorescences (F = 4.24, df = 1,54, P = 0.044). However, larger amounts of X. campestris were associated with faster bolting (Fig. 1B) and flowering (F = 5.9, df = 1,54, P = 0.018) times. In addition, the high dose of X. campestris resulted in fewer rosette leaves than the low dose (F = 5.7, df = 1,54, P = 0.020).
R Gene Resistance Can Limit Changes in Development with Infection
To evaluate whether R gene resistance alters developmental changes with infection, we compared the responses of RPS2 and rps2 plants to P. syringae + avrRpt2 and the responses of RPP5 and rpp5 plants to P. parasitica Noco2. For RPS2, we used wild-type Col in conjunction with an rps2 mutant in the experiment described in the first section. Significant Genotype × Infection Treatment interaction terms indicate that RPS2 resistance significantly altered flowering time, numbers of aerial and basal branches, and silique production by basal branches (Table IV). When infected, RPS2 plants took longer to flower, produced more aerial branches, fewer basal branches, and fewer siliques on basal branches than infected, susceptible plants (means contrasts, all P < 0.001). In each case, RPS2 plants infected with P. syringae more closely resembled mock-treated plants than rps2 plants and were only significantly different from mock-treated resistant plants for number of aerial branches (F = 5.45, df = 1,34, P < 0.05).
Table IV.
F ratios for interactions between resistance/susceptibility genotype, infection treatment, and competition treatment
Pathogen | Interaction | Flowering Time | Aerial Branches | Basal Branchesb | Siliques Produced by Basal Branchesb |
---|---|---|---|---|---|
P. syringae DC3000 + avrRpt2 | RPS2 genotype × infection | 31.9***a | 42.5*** | 5.42* | 37.0*** |
RPS2 genotype × infection × competition | 6.20* | 6.31* | |||
P. parasitica Noco2 | RPP5 genotype × infection | 1.20 | 0.72 | 0.04 | 0.19 |
RPP5 genotype × infection × competition | 1.36 | 0.59 |
a Asterisks denote significance of F ratio.
*, P < 0.05;
***, P < 0.001; F ratios are tested over line interaction terms.
b Traits measured only in the absence of competition.
For P. parasitica, we compared RPP5 lines and rpp5 lines that were created via the introgression of RPP5 from Landsburg erecta (Ler) into Col. The susceptible (rpp5) lines were homozygous for the Col RPP5 gene family, and resistant (RPP5) lines were homozygous for the Ler RPP5 gene family. In contrast to RPS2 resistance, RPP5 did not significantly alter plant responses to infection (Table IV). When infected, RPP5-resistant plants did not significantly differ from rpp5 plants in number of aerial branches, number of basal branches, or number of siliques on the basal branches (means contrasts, all P > 0.10). Although there were also no significant differences between infected and mock-treated RPP5 plants across competition treatments, in the presence of competition, RPP5 plants exhibited accelerated flowering with infection (F = 6.31, df = 1,34, P < 0.025).
One factor that might explain the difference in results for RPS2 and RPP5 resistance is the amount of pathogen present in resistant plants. Although both RPS2 and RPP5 resistance significantly reduced disease symptoms (data not shown), RPS2 resistance prevented visible disease symptoms almost entirely, whereas 65% of infected RPP5 resistant plants showed some sporulation by 11 d after infection. To investigate whether the amount of pathogen present might affect the development of plants with an R gene resistance response, we infected wild-type Col plants with three different doses of P. syringae DC3000 + avrRpt2, an incompatible strain. Here, P. syringae infection led to changes in the development of resistant plants, but only when the pathogen dose was sufficiently high (Fig. 1). Resistant plants infected with a low dose of P. syringae DC3000 + pLABL18 did not differ significantly from plants given the mock treatment for any of the developmental traits. Plants given medium and high doses produced significantly fewer secondary aerial meristems and aerial branches than mock-treated plants (both P < 0.001), and the numbers for both of these traits were significantly lower for the high dose than for the low dose (means contrasts, all P < 0.01). Like for compatible interactions with similar amounts of P. syringae in this experiment, there were no significant effects of the high and medium doses on time to bolting or flowering. The responses to the different doses of incompatible pathogen parallel differences in the amount of pathogen present after 4 d and symptoms of infection. High and medium doses resulted in significantly more pathogen present 4 d after infection than the low dose (means contrasts, both P < 0.01). The medium and high doses also caused visible hypersensitive response-induced cell death, whereas the low dose resulted in symptomless leaves (Fig. 1A).
DISCUSSION
In response to compatible pathogen infection, susceptible plants undergo localized chlorosis and cell death, increases in hormones such as SA, JA, ethylene, and auxin, and changes in the expression of transcription factors associated with stress and resistance (Dong, 1998; Maleck et al., 2000; Glazebrook, 2001; Chen et al., 2002; O'Donnell et al., 2003). Our experiments indicate that the susceptible response to pathogen infection also extends to changes in the timing of plant development and morphology. We found that P. syringae, P. parasitica, and X. campestris infections can each cause Arabidopsis plants to flower faster and produce fewer aerial branches. In addition, P. syringae infection and P. parasitica infection can increase basal branch development under high resource conditions. These results suggest that faster flowering times and changes in branch architecture are general consequences of pathogen infection in susceptible Arabidopsis seedlings. We propose that these developmental changes are initiated by the plant, are developmentally linked, and constitute a single developmental response to pathogen infection.
The changes in plant development could be either an active response of plants to infection or the consequence of pathogens actively manipulating plant growth. Some pathogens are known to alter plant growth by inducing hormonal changes (Agrios, 1997; Jameson, 2000), and DC3000 and other Pseudomonas pathovars have genes involved in auxin regulation (Glickmann et al., 1998). However, there are a couple of reasons why it is more likely that the changes in development are an active response of plants. First, the three kinds of pathogens we investigated are distantly related and have very different modes of infection, making it unlikely that these pathogens would manipulate Arabidopsis in similar ways. Second, the facts that plants respond to some stressful abiotic conditions with a faster transition to reproduction and that responses to abiotic and biotic stresses overlap, indicate that plants could initiate faster reproduction in response to pathogen infection.
The overlap between responses to abiotic factors and to pathogen infection suggests that some of the same pathways could be utilized to initiate accelerated flowering. The pathways responsible for inducing faster flowering in response to abiotic stresses are unknown. However, responses to some abiotic stresses, like to pathogen infection, involve increases in SA, JA, and ethylene (Wang et al., 2002), suggesting that one or more of these hormones could be involved in the accelerated flowering. Another interesting candidate for a role in the induction of the developmental changes is a change in auxin expression. O'Donnell et al. (2003) recently reported that auxin levels and auxin-regulated gene expression increase in Arabidopsis infected with a X. campestris strain or with P. syringae pv tomato, and suggest that the increase in auxin is probably initiated by the plant. Auxin is thought to coordinate developmental processes and is involved in photomorphogenesis, affects the positioning of organ development, and alters numbers of inflorescences (Reinhardt et al., 2000; Berleth and Sachs, 2001; Liscum and Reed, 2002; Reinhardt and Kuhlemeier, 2002).
Although the similarity of responses to different pathogens suggests that similar pathways are involved, the presence of some differences in susceptible plants' responses to the pathogens reveals that the processes differ in at least some details. In particular, similar amounts of P. syringae and X. campestris had different consequences for time to bolting and number of aerial branches (Fig. 1). In addition, the relationships between the amount of pathogen and time to bolting and between the amount of pathogen and number of rosette leaves differed between X. campestris and P. syringae. Further experiments are necessary to determine to what extent a common mechanism underlies the responses to different pathogens.
Our results suggest that developmental changes with infection are dependent on the presence of sufficient amounts of pathogen and/or disease symptoms and that R gene resistance can prevent developmental changes through reduced bacterial growth. When RPS2 resistance prevented symptoms and limited pathogen growth, developmental changes were absent or small. However, when RPS2-resistant plants exhibited a visible hypersensitive response, and high amounts of pathogen were present, developmental changes like those in susceptible plants occurred. This indicates that the presence of an R gene resistance response does not directly prevent the changes in development. In susceptible plants, changes in the number of secondary aerial meristems required sufficient pathogen and/or some visible disease symptoms (Fig. 1). General stress does not appear to be responsible for the changes in development in our experiments; plant rosettes did not appear purplish before flowering (personal observation), as can be the case for nutrient-stressed plants that flower faster.
The acceleration of flowering time and changes in branch architecture are likely to be developmentally linked. Faster flowering and the production of fewer aerial branches may both be a consequence of a transition to reproduction at an earlier vegetative stage (Diggle, 1999; Pidkowich et al., 1999). When there is such an early transition to reproduction, fewer leaves are produced. A reduction in leaf production may include the production of fewer cauline leaf primordia, all of which are produced before stem elongation (Hempel and Feldman, 1994). Because aerial meristems are produced in the axils of cauline leaves (Long and Barton, 2000), a reduction in the number of cauline leaves limits the opportunity for aerial branching. A developmental link between flowering time and the number of aerial branches is suggested by other studies. Often, mutations and light conditions that accelerate flowering time decrease the number of aerial branches, and mutations that slow flowering time increase the number of aerial branches (Martinez-Zapater et al., 1995; van Tienderen et al., 1996; Bradley et al., 1997; Pigliucci and Schmitt, 1999).
Although faster flowering sometimes coincided with less aerial branch production for P. syringae infection (Table I; Fig. 2), it did not always. For example, plants infected with large amounts of P. syringae produced fewer aerial branches but did not flower faster (Figs. 1 and 2). One possible explanation for this pattern is that extensive P. syringae disease symptoms slow plant growth and prevent faster flowering while still permitting a transition at a relatively early vegetative stage. In addition, we observed that very low titers of P. syringae, which caused little or no visible symptoms, resulted in faster flowering without decreases in the number of aerial meristems (Fig. 1). This suggests that faster flowering with pathogen infection may also occur by mechanisms other than a switch to reproduction after fewer cauline leaf primordia have been produced.
The smaller number of secondary aerial meristems with pathogen infection may cause the increase in basal branching under high resource conditions. After the transition to flowering, secondary meristem development is basipetal, with the uppermost secondary meristem producing the first branch (Hempel and Feldman, 1994). When there are fewer aerial meristems, the release of meristems may proceed more readily down to meristems in the axils of rosette leaves (Sell, 1980), leading to more basal branches. The increase in basal branching we observed resembles the regrowth response of some plants to herbivory. However, with herbivore damage, the increased basal branching is due to a loss of apical dominance from clipping of the primary inflorescence (Tiffin, 2000); in our experiments, there was no visible damage to the inflorescences.
The developmental changes with infection might enhance a plant's ability to cope with pathogen infection in several ways. First, faster development may lead to the early development of age-related resistance in Arabidopsis (Leisner et al., 1993; Martin et al., 1997; Kus et al., 2002). Second, some of the developmental changes with infection may increase early seed production, which could enhance the fitness of plants that may be prematurely killed by the pathogen. Faster flowering increases the likelihood that plants will be able to produce some seed before succumbing to disease (Hochberg, 1992; Agnew et al., 2000). Finally, increases in basal branching may enhance a plant's ability to compensate for losses due to pathogen infection. Increased basal branching in response to herbivore damage can compensate for loss of the primary inflorescence meristems (Inouye, 1982; Paige and Whitham, 1987; Doak, 1991). With pathogen infection, the increases in the number of basal branches may compensate for the production of fewer aerial branches. Further work is necessary to determine if the developmental response to pathogen infection that we observe enhances plants' abilities to escape from, resist, or compensate for disease and what molecular pathways are involved in the developmental responses.
MATERIALS AND METHODS
Plant and Pathogen Materials
RPS2-resistant and -susceptible lines were made from wild-type Arabidopsis Col seed, provided by Jean Greenberg (University of Chicago, IL), and Col rps2-101C seed (Mindrinos et al., 1994), provided by Fred Ausubel (Harvard University, Cambridge, MA). RPP5-resistant and -susceptible lines were made from Y209, a Col × Landsberg F2 (Parker et al., 1997) provided by Jane E. Parker (Max-Planck-Institute for Plant Breeding Research, Koln, Germany), and Col, obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus).
Experiments utilized the following Pseudomonas strains: Pseudomonas syringae pv tomato strain DC3000 (Whalen et al., 1991), P. syringae pv maulicola strain ES4326 (provided by Jean Greenberg), P. syringae strain RM18.1 (Jakob et al., 2002), and P. syringae pv phaseolicola strain NPS3121 (provided by Jean Greenberg). Peronospora parasitica strain Noco2 was provided by John M. McDowell (Virginia Tech, Blacksburg, VA), and Xanthomonas campestris pv campestris strain 8004 was provided by Jeff Dangl (University of North Carolina, Chapel Hill, NC).
Growth Conditions and Inoculation Methods
Plants were grown in Promix soil in a growth room (300 μmol photons m-2 s-1) with a 12-h-light/12-h-dark cycle. Planted seeds were given a 2- to 5-d cold treatment at 4°C. Plants were not fertilized, and pots were rotated every 1 or 2 d to adjust for uneven lighting across the growth room. Due to different growth requirements for different pathogen strains, some conditions varied among experiments and are noted with the methods for each experiment.
In all experiments, the first two true leaves were infected when plants were approximately 2 weeks old. For bacterial infections, P. syringae strains were grown in King's Broth media (King et al., 1954) at 28°C to 30°C, and the X. campestris strain was grown in Luria-Bertani media at 30°C to mid-log phase. Cultures were spun in a microcentrifuge at 3,000 rpm for 5 min, washed one time in 10 mm MgSO4, and resuspended in 10 mm MgSO4 to the appropriate bacterial concentration using a spectrophotometer. Bacterial solutions were infiltrated into the bottom surface of leaves using a blunt-ended syringe. The mock treatment entailed infiltration of the buffer, 10 mm MgSO4.
For P. parasitica infection, plants were infected with asexual conidiospores harvested from sporulating seedlings that had been inoculated 1 week before the experiment. Sporulating seedlings were shaken in water, the solution was spun, and the condiospores were resuspended. The concentration was adjusted to 3.5 × 105 zoospores mL-1 using a hemocytometer. Five microliters of the spore suspension was dropped onto each leaf with a pipette. The mock treatment entailed pipetting water. Because P. parasitica requires high humidity for infection, pots were placed within polyvinyl chloride frames covered with Warp's polycomb plastic sheeting. Before P. parasitica infection, pots were transferred to flats with wet sponge bottoms and, to prevent transmission of the P. parasitica to other plants, were bottom watered for a few weeks after infection.
Effect of P. syringae pv tomato DC3000 on Susceptible and Resistant Plant Development
To create homozygous resistant and susceptible lines that had the same genetic background and parental history, rps2-101C was crossed to wild-type Col, and the progeny of a single heterozygous parent were screened for homozygous RPS2 and rps2 plants using a cleaved-amplified polymorphic sequence (CAPS) marker. The selfed progeny of each selected homozygous plant were used as a line; 18 resistant and 18 susceptible lines were used.
Plants were infected with either P. syringae pv tomato DC3000 containing the avrRpt2-bearing vector pLABL18 (Whalen et al., 1991) at OD600 = 0.0001 or a mock treatment of 10 mm MgSO4. Plants were grown in 12.5-× 10-× 10-cm pots at 20°C with domes for 1 d after infection. In the competition treatment, six wild-type Col plants were planted 3 cm away from the focal plant in an evenly spaced circle around the focal plant. Only the focal plant was infected. In the no-competition treatment, there was only a focal plant in each pot. There were two replicates of each line for each combination of infection type and competition level. All pots were randomly positioned in the growth room.
Numbers of aerial branches, basal branches, siliques on the basal branches, and height were determined after senescence. Each trait was analyzed with an ANOVA using Statistica. Line was considered a random effect and competition treatment, infection treatment, and resistance genotype were considered fixed effects. The statistical significance of effects of pathogen versus mock treatment for susceptible plants was evaluated using means contrasts across competition treatments and within competition treatments. Because basal branches were rare in the presence of competition, ANOVAs were done for number of basal branches and number of basal siliques for the no competition treatment only. To determine the effect of RPS2 resistance, we evaluated the significance of the interaction terms Infection Treatment × Resistance Genotype and Infection Treatment × Resistance Genotype × Competition. In addition, to evaluate the nature of these interactions, means contrasts were calculated between infected, susceptible and resistant plants and between infected and uninfected resistant plants.
Effects of P. parasitica Noco2 on Susceptible and Resistant Plant Development
Near-isogenic lines were made by introgressing the Ler RPP5 gene family into Col, which is susceptible to P. parasitica Noco2 (Parker et al., 1993). A Col × Ler F2 line containing RPP5, Y209 (Parker et al., 1997), which has a recombination point approximately 90 kb away from RPP5 on the centromeric side, was backcrossed to Col six times to create F7 plants. In each generation, progeny from the backcrossed siliques were screened for the presence of RPP5 using a CAPS marker that was created based on differences between the RPP5 alleles. To reduce the size of the introgressed segment, a plant with recombination points close to RPP5 was selected with a CAPS marker 245 kb away from RPP5 on the telomeric side. A single F7 heterozygote was selfed, and its progeny were screened for homozygous RPP5 and rpp5 plants with a CAPS marker. Each selected homozygous plant was selfed to create a line; 18 resistant and 18 susceptible lines were created. The RPP5 resistance phenotype was confirmed for a subset of the lines by infecting 1-week-old seedlings with P. parasitica and counting the number of seedlings with sporulation.
The experiment entailed the same conditions, experimental design, measurements, and analyses described above for P. syringae pv tomato DC3000. Because many plants had no basal branches, there was a non-normal distribution for number of basal siliques. Thus, differences in the number of siliques on the basal branches were analyzed using line means in an ANOVA.
Effects of X. campestris and Amounts of Compatible and Incompatible P. syringae
Plants were infected with four strains of P. syringae: P. syringae pv tomato strain DC3000, P. syringae pv maulicola strain ES4326, P. syringae strain RM18.1, and P. syringae pv phaseolicola strain NPS3121, at three initial doses, OD600= 0.03, 0.003, and 0.0003. In addition, control plants were treated with a mock treatment consisting of 10 mm MgSO4. Plants were grown at 18°C in 6-× 9-× 9-cm pots, and domes were placed over the flats for 3 d after infection. The amount of bacteria 4 d after infection was measured in separate plants from those used to measure the effect of infection on development. For each strain dose combination, one whole infected leaf was clipped from each of six plants and ground in 10 mm MgSO4. Dilutions were plated on King's Broth plates with a Whitley Automatic Spiral Plater. The number of colony-forming units per milliliter was determined by counting colonies with a ProtoCol colony counter (Synbiosis, Frederick, MD).
For measurements of development, 19 plants were infected for each pathogen dose treatment or the mock treatment. Disease symptoms were measured 9 d after infection by blindly scoring the percentage of infected leaf area with visible chlorosis, water soaking, or cell death. Average log pathogen abundance 4 d after infection and the average of each plant trait were calculated for each treatment combination. These were used to calculate regression coefficients between values for the traits and pathogen abundance.
Additional Col plants were infected with an incompatible strain, P. syringae DC3000 + pLABL18 (Whalen et al., 1991), which carries avrRpt2, at the same three doses. To determine if any of these doses altered development, one-way ANOVAs with the factor Treatment, which included the three doses and mock treatment, were calculated. When Treatment had a significant effect, means contrasts were used to determine the significance of differences between the mock treatment and each of the doses. To investigate whether amount of P. syringae DC3000 + pLABL18 affected development, one-way ANOVAs that included Dose (without mock treatment) were performed, and a means contrast was calculated between the high and low dose.
Additional plants were infected with X. campestris pv campestris strain 8004 (obtained from Jeff Dangl) at the same three doses. The amount of pathogen 4 d after infection was determined by plating dilutions of ground leaves on Luria-Bertani plates. The same analyses as for P. syringae DC3000 + avrRpt2 were performed. For analyses of the effects of X. campestris presented in Tables II and III, means contrasts were calculated between the high dose and the mock treatment. The high dose was chosen because the amount of pathogen after 4 d for this dose was closest to that of P. syringae in the first experiment.
To investigate the effect of amount of compatible P. syringae on basal branching, a separate experiment was done in 12.5-× 10-× 10-cm pots at 20°C with domes on the flats for 1 d after infection. Col rps2 plants were infected with three doses of P. syringae DC3000 + pLABL18 (carrying avrRpt2) or given a mock treatment. The same analyses were done as for P. syringae DC3000 + pLABL18 in the experiment with smaller pots. Unlike in the other experiments, plants were not rotated among all positions; as a consequence, the ANOVAs included row as a blocking factor. Because there was no replication within rows, Row × Dose served as the error term.
Distribution of Materials
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
Acknowledgments
We thank Jean Greenberg for advice and comments on this manuscript and the V. Dropkin Foundation for the use of equipment.
This work was supported by the National Science Foundation (Dissertation Improvement Grant to T.M.K.) and by the National Institutes of Health (grant no. GM57994 to J.B.).
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