Life cycle stages of Peronospora parasitica in wild-type Arabidopsis. Blue perimeter circle shows approximate time when each development stage appears in wild-type hosts (dai: days after inoculation). Interior black numbers correspond to the developmental stages listed in Table 1. At 0 – 1 dai, conidiospores germinate and form germ tubes ( A, two fields shown; stage 1), which penetrate the host epidermis and deposit primary haustoria into plant cells at the sites of ingress ( B ; stage 2 – 3). Vegetative growth produces small colonies of hyphae adorned with haustoria ( C and D ; stage 4), which proliferate to form large colonies within the leaf ( E and F ; stage 5). Stages 6 – 8 include differentiation of antheridia and oogonia, sexual union, and formation of diploid oospores ( H and I ). At stage 9, conidiophores are formed that emerge through stomata and produce vegetative spores or conidia ( G ). c = conidiophore; gt = germ tube; h = hyphae; ha = haustorium; o = oospore; pha = primary haustorium; t = trichome (plant). Scale bars = 25 μM for A, B, and I ; 50 μM for D and H ; 100 μM for C, E, and G ; 2 mm for F . 

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... Delaney et al. 1995; Glazebrook et al. 1996; Shah et al. 1997). Following infection, nim1/npr1 mutants show normal pathogen-induced accumulation of SA but greatly reduced induction of SAR-associated genes such as PR-1. Additionally, exoge- nous application of SA or INA to nim1/npr1 plants neither induces SAR genes nor activates resistance to pathogens, as would occur in wild-type plants. Therefore, the NIM1/NPR1 gene product acts to couple accumulation of SA to the activation of defense genes and resistance. Homozygous nim1/npr1 plants and NahG plants are similar in that each can be considered “immune compro- mised,” with respect to SAR induction. NahG plants, however, exhibit a more severe defense-impaired phenotype than nim1/npr1 mutants, indicating that SA may control other resistance mechanisms beyond those dependent upon the NIM1/NPR1 pathway. It also has been speculated that the hypersusceptible phenotype of NahG plants may result in part from altered feedback regulation of the phenylpro- panoid pathway brought on by SA depletion. It was suggested that this may lead to changes in the synthesis of other products of this pathway and less resistance to disease (Cameron 2000). To understand the complexity of SA- mediated resistance mechanisms will require careful examination of the biochemical and defense phenotypes of wild- type, NahG, and nim1/npr1 plants. Immune-compromised plants also can be important sub- strates on which to observe the growth and development of compatible pathogens with less interference from induced host defenses. This is illustrated by the accentuated virulence displayed by a wide range of pathogens on NahG tobacco and Arabidopsis plants compared with the wild type, showing that even susceptible hosts for phytopathogens exert defense functions that reduce pathogen fitness and constrain disease severity (Delaney et al. 1994). Furthermore, though wild-type hosts may recover from infection by compatible pathogens, nim1 and NahG Arabidopsis plants often succumb and die, most likely as a result of a failure in activation of induced defense responses (H. Kim and T. Delaney, unpublished data ). These observations underscore the differences between wild-type plants and those with compromised immune systems, with respect to their suitability as hosts for virulent pathogens. To assess the roles played by SA and NIM1/NPR1- dependent processes on pathogen virulence, we examined the growth and development of a virulent P. parasitica isolate on A. thaliana wild-type and immune-compromised NahG and nim1-1 plants. To facilitate comparisons, all host genotypes were derived from the same A. thaliana accession (Ws-0), which were inoculated with the virulent P. parasitica isolate Emwa. We dissected the pathogen’s life cycle into nine stages, many of which were documented and quantified in each host genotype. We found P. parasitica to show the greatest fitness enhancement over wild type in NahG, followed by nim1-1 plants, on the basis of hyphal development, pathogen rRNA accumulation, and production of conidiophores. To gain insight into the basis for enhanced pathogen growth on NahG and nim1-1 plants, we measured accumulation of mRNA from the SAR marker gene PR-1 and characterized the abundance and morphology of callose deposits that surround haustoria. Compared with the wild type, NahG and nim1-1 plants showed much less PR-1 accumulation, as previously observed (Delaney et al. 1994; Delaney et al. 1995). Four types of extrahaustorial callose deposits were defined, and their frequency was assessed in each host genotype. Interestingly, significant differences between host genotypes were apparent with respect to the abundance of callose deposits in the form of collars or thick encasements around haustoria. Application of INA to already infected NahG plants converted the callose profile in these plants to closely resemble the wild-type pattern, com- mensurate with substantial accumulation of PR-1 mRNA. In contrast, INA had no significant affect on the callose profile of nim1-1 plants and caused only a small increase in PR-1 mRNA. Together these results suggest that extrahaustorial callose production is enhanced by treatments that activate SAR, and that this induction may involve the NIM1/NPR1 pathway. To characterize the growth and development of a virulent Peronospora isolate (Emwa1) on wild-type Arabidopsis, we placed 2.0 μl drops of water containing approximately 50 P. parasitica conidiospores onto young leaves of susceptible ecotype Ws-0. Leaves were harvested every 2 h postinoculation (hpi) for the first 12 h and every 24 hpi for the next 6 days. Leaf samples were fixed and stained in lactophenol trypan blue, cleared in chloral hydrate, and observed by microscopy to visualize pathogen structures (Uknes et al. 1993). Trypan blue is commonly used to stain necrotic plant cells and fungal tissues that are living at the time of fixation. We dissected the P. parasitica life cycle into nine stages (Fig. 1) and recorded the approximate timing of their occurrence in wild- type, nim1-1 , and NahG plants (Table 1). By 2 hpi, approximately 25% of the conidiospores had germinated and many had already formed appressoria over the juncture of adjoining epidermal cells, a pattern described previously (data not shown) (Chou 1970; Koch and Slusarenko 1990). The germination frequency did not vary appreciably between host genotypes and was typical of what we normally observe for P. parasitica on Arabidopsis. We noticed that conidia landing close to a juncture between adjacent epidermal cells would form an appressorium di- rectly, without a noticeable germ tube. Spores more distant from a juncture, however, would produce conspicuous germ tubes (typical is 7 to 11 μM) (Fig. 1A) that would extend until a juncture was reached. Germinated spores showed little trypan blue staining, whereas their germ tubes and appressoria stained darkly. This staining pattern may reflect the cytoplasmic contents moving out of the spore into newly formed structures in preparation for penetration (Chou 1970; Mendgen and Deising 1993). From 4 and 8 hpi, the pathogen had not progressed beyond appressorium formation, but by 10 to 12 h, penetration of the leaf surface had occurred and one or two epidermal cells flanking the penetration site had been invaded by haustoria (Fig. 1B), similar to that described by Koch and Slusarenko (1990). In some instances, the developing hyphae had invaded the mesophyll by 12 h. At 1 day postinoculation (dpi), small colonies were established in the mesophyll layer. At this time, the infection site usually showed dark trypan blue staining caused by heavier staining of primary haustoria in the epidermal cells and in the first several mesophyll cells colonized. These haustoria usually were larger than those formed later. By day 2, a typical colony in each of the host genotypes was composed of a nonbranched hypha with approximately 70 to 90 haustoria growing within the mesophyll (Fig. 1C). Nascent haustoria were apparent near hyphal tips as small, round pro- tuberances, whereas mature haustoria that were less proximal to the apex appeared larger and lobular (Fig. 1D and I). After day 2, intercellular hyphae continued to colonize the leaf mesophyll, forming frequent dichotomous branches and dif- ferentiating usually by one (Fig. 1D to F), but occasionally as many as three haustoria per host cell. As early as 3 dpi, we observed regions of hyphal swelling, which stained more darkly than other hyphal sections. We attributed these swellings to production of oogonial initials as described by Dick (1994). In support of this interpretation, by day 4 mature oogonia were numerous, septate, and usually had paragynous antheridia appressed to their sides. By 5 dpi, mature, aplerotic oospores were present, which were heavily stained (Fig. 1H and I). We did not observe oospores to gener- ate secondary vegetative hyphae, although occasionally we did notice that by 6 dpi, oospores had apiculus-like structures, suggesting that secondary somatic growth may occur in this system, as observed in other oomycetes (data not shown) (Dick 1994). At 4 to 5 dpi, conidiophore initials were apparent as thick- ened hyphal sections under the epidermis near stomatal openings. Usually one, but occasionally multiple conidiophores would emerge through a single stomatal opening. By day 5, these had differentiated into mature conidiophores that were roughly the size of a leaf trichome, with branches bearing terminal conidiospores (Fig. 1G). By 6 dpi, infected leaves contained an elaborate hyphal net- work that deposited haustoria into approximately three quarters of the adjacent mesophyll cells. Leaves contained numerous oospores, and their surfaces were decorated with a prominent flush of hygroscopic conidiophores that would twist in the air currents, releasing clouds of conidiospores. At this time, sexual and asexual life cycles of the pathogen were complete and, although abundant host tissue was colonized, most plant cells appeared healthy, as visualized by the lack of trypan blue staining. Leaves formed later than 1 week after infection were free of the pathogen, allowing wild-type plants to outgrow the disease. To compare growth and development of P. parasitica on wild-type and immune-compromised plants, we inoculated each host genotype and monitored parasite development, as described above, focusing on the timing of life-cycle events and the amount of pathogen structures formed (Table 1). Our goal was to determine which pathogen life-cycle stages were most affected by SA or NIM1/NPR1-dependent processes, phases that should show the greatest alteration in the immune- compromised plants in our study. We analyzed early pathogen growth stages up to 2 dpi, but saw no significant differences among host genotypes in spore germination or penetration efficiency, speed of colony establishment, or frequency of haustoria formation (data not shown). By ...