Differences in early callose deposition during adapted and non-adapted powdery mildew infection of resistant Arabidopsis lines

Abstract

The deposition of callose, a (1,3)-β-glucan cell wall polymer, can play an essential role in the defense response to invading pathogens. We could recently show that Arabidopsis thaliana lines with an overexpression of the callose synthase gene PMR4 gained complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum and the non-adapted powdery mildew Blumeria graminis f. sp hordei. The penetration resistance is based on the transport of the callose synthase PMR4 to the site of attempted fungal penetration and the subsequent formation of enlarged callose deposits. The deposits differed in their total diameter comparing both types of powdery mildew infection. In this study, further characterization of these callose deposits revealed that size differences were especially pronounced in the core region of the deposits. This suggests that specific, pathogen-dependent factors exist, which might regulate callose synthase transport to the core region of forming deposits.

Keywords: :

• (1,3)-β-glucan
• Arabidopsis thaliana
• AtGSL5
• PMR4
• callose synthase
• cell wall
• papillae
• plant defense
• powdery mildew

The (1–3)-β-glucan, callose, is involved in multiple aspects of cell well modification during plant growth, development and in response to stress.^1-4An early defense response to invading pathogens in plants is the formation of so called papillae, which are cell wall thickenings with callose as the most prominent constituent.⁵These papillae are thought to act as a physical barrier to prevent or slow pathogen penetration.⁶ The plant can gain time to activate an internal cascade of biological processes necessary for subsequent defense responses^7,8 and global transcriptional changes.⁹

In our recent study, we could directly confirm that callose formation can function as barrier to prevent fungal penetration. Arabidopsis (Arabidopsis thaliana) lines that overexpressed the callose synthase gene PMR4 showed complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum (Gc) and the non-adapted powdery mildew Blumeria graminis f.sp hordei (Bgh).¹⁰ The callose synthase PMR4 (also called GSL5) is responsible for stress-induced callose deposition,^2,11 which was identified in pmr4 disruption mutants. Although pmr4 disruption mutants did not deposit callose at sites of attempted fungal penetration, they unexpectedly revealed an increased resistance to powdery mildew. A double mutant and microarray analyses showed that the hyperactivated salicylic acid pathway is causative for the high resistance in the pmr4 mutant.¹¹ However, the resistance of the PMR4 overexpression lines 35S::PMR4-GFP was independent of an hyperactivated salicylic acid pathway but based on an elevated early callose deposition at the site of attempted powdery mildew penetration.¹²Callose deposits were significantly enlarged compared with wild type during both, Gc and Bgh infection. However, comparing the size of callose deposits within the resistant 35S::PMR4-GFP line, especially the diameter of the first callose deposit induced by Gc infection was significantly larger than that of the Bgh-induced deposit.¹²

In this study, we further characterized differences of callose deposits induced at early time-points of Gc and Bgh infection in the resistant 35S::PMR4-GFP lines and wild type. The special focus was on the size difference of the core region of the first and second callose deposit. Three-week-old 35S::PMR4-GFP lines and wild-type plants were inoculated with Gc and Bgh as previously described.¹² Rosette leaves were taken 6, 12 and 24 hpi and stained with aniline blue to visualize callose deposition by fluorescence microscopy.^2,10,11 At 6 hpi with Gc, the first callose deposit was fully established (Fig. 1A).¹²Whereas callose deposits of the resistant 35S::PMR4-GFP lines showed a central callose core region surrounded by a field of callose, the callose deposit had a dot-like shape without a distinct core in wild type (Fig. 1A). Comparing Gc and Bgh infection of 35S::PMR4-GFP lines, we determined a 3 times larger diameter of the core region of Gc-induced deposit 6 hpi (Fig. 1B). A first callose deposit was not formed during Bgh infection of wild-type leaves (Fig. 1A).¹⁰ A difference in the core diameter was not observable 12 hpi with the adapted and non-adapted powdery mildew (Fig. 1B); but at 24 hpi with Bgh, the diameter of the callose core in 35S::PMR4-GFP lines decreased to the size that was detected at 6 hpi, while the core diameter of the first callose deposit remained stable during Gc infection (Fig. 1A). In wild type, the core region like the whole callose deposit was diffuse,¹⁰ which did not allow a determination of the diameter.

Figure 1. Callose deposition and callose synthase localization in the core region of callosic papillae in Arabidopsis during powdery mildew infection.Three-week-old resistant 35S::PMR4-GFP lines and wild type were inoculated with the adapted powdery mildew G. cichoracearum (Gc) and the non-adapted powdery mildew B. graminis f.sp hordei (Bgh). All tests were conducted with rosette leaves. (A) Micrographs showing callose deposition (blue fluorescence by aniline blue staining) at sites of attempted fungal penetration at 6 and 12 hpi. Conidia washed off (except Bgh infection of wild type at 6 hpi) to improve visualization of callose deposits. 1: first callose deposit, 2: second callose deposit. C, conidium; pp, penetration peg. Scale bar = 10 µm. (B) Diameter of the core region of the first [at primary (Bgh) and appressorial (Gc) germ tube, respectively] and second callose deposit [at appressorial germ tube (Gc + Bgh)] in aniline-blue stained leaves 6, 12 and 24 hpi. nd: not detectable. *p < 0.05, **p < 0.01 Tukey's test. Error bars represent ± SEM, and n = 100 of 4 independent leaves. (C) Ratio of total diameter of callose deposits¹² vs. diameter of the core region (this study). (D) Z-projected confocal laser-scanning micrographs at sites of attempted fungal penetration. Conidia were stained with propidium iodide. Arrow indicates GFP fluorescence emitted from PMR4-GFP fusion protein at the site of the first (1) and second (2) callose deposit. Micrographs are representative for 12 hpi. Agt, appressorial germ tube; c, conidium; pgt, primary germ tube. Scale bar = 10 µm.

We observed similar differences in the core size at the second callose deposit that was fully established at 12 hpi.¹²At this time-point, the core diameter of the second callose deposit induced by Gc infection in 35S::PMR4-GFP lines was 4.5 times larger compared with the Bgh induced core region, but was not significantly different to the Bgh induced core region in wild type (Fig. 1B), which, however, revealed a penetration peg (Fig. 1A). At this time point during Gc infection, not a distinct core region but a central penetration peg was formed in the second deposits of wild-type leaves (Fig. 1A). We also determined a larger size of the core region of the second callose deposit in Gc-infected epidermal leaf cells than in Bgh-infected cells of the 35S::PMR4-GFP lines at 24 hpi, however, the size difference was reduced to factor of 1.5 (Fig. 1A). In wild type, secondary callose deposits revealed fully established penetration pegs in the central region at 24 hpi with Gc and Bgh (Fig. 1B).¹⁰

The differences in the formation of the callose deposit, that especially affected the size of the core region, are also reflected in the ratio of the total diameter of callose deposits¹² to the diameter of the core region (Fig. 1B). In 35S::PMR4-GFP lines, the ratio for callose deposits after Gc infection was between 2.6 and 3.4, whereas a ratio of 4.1 to 5.2 was calculated for Bgh-induced callose deposits, except the second callose deposit at 24 hpi with a ratio of 2.7 (Fig. 1C). In wild type, the calculation of a ratio was only possible for the first callose deposit at 12 hpi infection with Gc and for the second callose deposit at 12 hpi with Bgh because these were the only time points with a distinct core formation in callose deposits (Fig. 1B). The ratio for Gc-induced first callose deposits at 12 hpi in wild type was very similar to the ratio of deposits in 35S::PMR4-GFP lines (Fig. 1C). Contrarily, the ratio for Bgh-induced secondary callose deposit at 12 hpi in wild type (ratio: 1.8) was far below the ratio in 35S::PMR4-GFP lines (ratio: 4.1) (Fig. 1C). Based on these results, we conclude that not only the amount of callose deposited at the site of attempted fungal penetration is important for penetration resistance but also the structure of the deposit. Besides an overall increased callose deposit, a strongly enlarged core region might be required to induce penetration resistance to an adapted powdery mildew like Gc in the interaction with Arabidopsis. Hence, differences in the regulation of callose formation seem to be responsible for the specific callosic patterns comparing wild type and resistant lines but also different types of powdery mildew infection, like Gc and Bgh.

In our recent study, we identified the transport of the callose synthase PMR4 from the plasma membrane to the site of infection via vesicle-like bodies as a main regulatory mechanism of stress-induced callose formation.¹²Differences in the transport of PMR4 during Gc and Bgh infection could also be responsible for the observed differences in the shape of the callose deposits and especially the core region. Confocal laser-scanning microscopy revealed that the differences in callose deposit formation are likely based on differences in the accumulation of the callose synthase PMR4. Whereas the GFP signal of the tagged PMR4 resembled the shape of the callose deposit, a central core with a surrounding field, at 12 hpi with Gc, a structural accumulation of PMR4 was not observed during Bgh infection (Fig. 1D). This suggests that apart from a general signal for the translocation of PMR4 to the site of attempted penetration additional regulatory factors are induced and activated for the specific localization of PMR4 at the site of fungal penetration during compatible and incompatible interactions. These factors might interact with the vesicle-like bodies that transport the callose synthase PMR4¹⁰ and regulate the positioning of the PMR4 bodies at sites of attempted penetration resulting in the specific localization of the callose synthase (Fig. 1D). Because PMR4 was present in the SYP61 trans-Golgi network compartment,¹³ these regulatory factors might play a role in exocytic trafficking, which could be also related to exosome transport, a possible mechanism of PMR4 translocation.^{10, 13} What these putative regulatory factors are and whether they might be involved the positioning of vesicle-like bodies that contain the callose synthase PMR4 still has to be elucidated.

Abbreviations:
PMR4	=	Powdery mildew resistant 4
GSL5	=	Glucan synthase like 5
hpi	=	hours post-inoculation
GFP	=	green fluorescent protein

Acknowledgments

Funding was provided in part by a postdoctoral research fellowship from the Deutsche Forschungsgemeinschaft (C.A.V.), the German Federal Ministry of Education and Research (BMBF, FKZ 0315521A, C.A.V., M.N.), the Carnegie Institution of Science (S.C.S.) and the Energy Biosciences Institute (S.C.S. and C.A.V.).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.