Detection and Localization of PIG Products

4.9.1 In vitro and in vivo Detection of PIG Products

To detect the presence of the remaining PIG-proteins (PIGp 5, 7, 14, 15) in the different development stages, proteins from in vitro and in vivo infection structures were isolated and analyzed by Western blots (see chapter 3.9.4). In addition, to positively verify the existence and function of the predicted secretion signal in each of the above PIGps (see chapter 4.2.1), it was tested whether yeast would recognize these signals and secrete PIGp into the growth medium. For this test, each of the four PIGs was expressed in the heterologous system Saccharomyces cerevisiae using the yeast vector pDR195. Proteins from the resulting yeast pellets and supernatants were isolated and - in some cases – deglycosylated, and submitted to Western blotting, together with the abovementioned proteins from in vivo infection structures.

All Western blots were carried out using polyclonal antibodies against the respective PIGp.

On the following pages, the Western blot results for the PIG-proteins in haustoria, in in vitro structures, as well as in the heterologous expression systems of E. coli and S. cerevisiae, are described.

In Figure 4-9 below, the Western blot for PIG7p detection in haustoria and heterologous expression are shown:

Figure 4-9 Protein detection by Western blot using polyclonal antibodies against PIG7p (RTP1p). The lanes are labeled as follows: YP7: pellet of S. cerevisiae:pDR195:PIG7 (20µg), YPC: pellet of S. cerevisiae:pDR195 (20µg), SY7: supernatant of S.

cerevisiae:pDR195:PIG7 (5µg), SYC: supernatant of S. cerevisiae:pDR195 (5µg), SY7d:

deglycosylated supernatant of S. cerevisiae:pDR195:PIG7 (5µg), SYCd: supernatant of S. cerevisiae:pDR195, FP7: fusion protein PIG7p (antigen) (0.1µg), H: haustorial protein extract

Four distinctive antibody cross-reaction signals (at ca. 25, 32, 80 and 90kDa) are visible in lane YP7, where protein extract from the PIG7-transformed yeast pellet was applied. The protein extracts from the non-deglycosylated supernatant (lane SY7) displays one strong signal (at about 100 kDa) that is most likely a combination of two signals similar to the upper two signals in lane YP7. Lane SY7d with the deglycosylated supernatant of the transformed yeast contains one strong signal at ca. 22 kDa. Thus, it can be concluded that the yeast S. cerevisiae recognizes the secretion signal of PIG7p.

In addition, we see that in contrast to the protein produced in yeast (lane SY7), the protein extract from haustoria (lane H) shows two smaller antibody cross reactivity signals at ca. 23.5 kDa for and at ca. 25.0 kDa. The smaller protein may be explained as a PIG7p pre-protein or a non-glycosylated form of the assumed mature protein. The signal for a larger protein in lane SY7 is due to the fact that the resulting heterologous protein in yeast is hyper-glycosylated compared to the original protein produced by the rust fungus in the haustorium.

The Western blotting results for PIG7p in vitro detection in are not shown, as no clear antibody cross-reactivity was detected in the U. fabae spores, and no antibody cross-reactivity at all were detected in U. fabae in vitro infection structures (2h, 4h, 20h, 24h) using antibodies against PIG7p.

Next, in Figure 4-10 below, the Western blot for PIG14p detection in haustoria and in heterologous expression systems is shown carried out with proteins extracted from transformed yeast, E. coli and haustoria:

Figure 4-10 Protein detection by Western blot using polyclonal antibodies against PIG14p. The lanes are labeled as follows: YP14: pellet of S. cerevisiae:pDR195:PIG14, YPC: pellet of S. cerevisiae:pDR195, SY14: supernatant of S. cerevisiae:pDR195:PIG14, SYC: supernatant of S. cerevisiae:pDR195, SY14d: deglycosylated supernatant of S.

cerevisiae:pDR195:PIG14, SYCd: supernatant of S. cerevisiae:pDR195, FP14: fusion protein PIG14 (antigen) (expressed in E. coli), H: haustorial protein extract

Two distinctive antibody cross-reactivity signals (at ca. 29 and 32 kDa) are visible in lane YP14, where protein extract from the PIG14-transformed yeast pellet was applied. The protein extract from the supernatant (lane SY14) shows one signal at ca. 120 kDa. In lane SY14d with the deglycosylated supernatant of the transformed yeast, one signal can be seen at 29 kDa. The conclusion that follows from these results is that the yeast S. cerevisiae recognizes the secretion signal of PIG14p.

In contrast to the protein produced in yeast (lane SY14), the protein extract from haustoria (lane H) shows one weak antibody cross-reaction signal at 29 kDa. As with PIG7p, this is due to the fact that the heterologous protein in yeast is more strongly deglycosylated than the original protein produced by the rust fungus in the haustorium.

The Western blot for PIG14p in vitro detection in are not shown, as no clear antibody cross-reaction signals were detected in the U. fabae spores, and no signals at all were detected in U.

fabae in vitro infection structures (2h, 4h, 20h, 24h) using antibodies against PIG14p.

To positively verify the function of the predicted secretion signal in PIG15p, it was tested whether yeast would recognize this signal and secrete PIG15p into the growth medium. The results of the Western blot for PIG15p, carried out with protein extracts from transformed yeast, E. coli, as well as from haustoria, can be seen in Figure 4-11 below.

Figure 4-11 Protein detection by Western blot using polyclonal antibodies against PIG15p. The lanes are labeled as follows: YP15: pellet of S. cerevisiae:pDR195:PIG15, YPC: pellet of S. cerevisiae:pDR195, SY15: supernatant of S. cerevisiae:pDR195:PIG15, SYC: supernatant of S. cerevisiae:pDR195, SY15d: deglycosylated supernatant of S.

cerevisiae:pDR195:PIG15, SYCd: supernatant of S. cerevisiae:pDR195, FP15: fusion protein PIG15p (antigen), H: haustorial protein extract

Two distinctive antibody cross-reaction signals (at about 29 and 32 kDa) are visible in the lane YP15, where protein extract from the PIG15-transformed yeast pellet was applied.

Unexpectedly, two antibody cross-reaction signals (at about 45 kDa and 47 kDa) also occur in the lane YPC (control), were protein extract from the vector-transformed yeast was applied.

The protein extract from the supernatant (lane SY15) displays no signal, as does the deglycosylated supernatant of the transformed yeast (lane SY15d). The protein extract from haustoria (lane H) shows one clear antibody cross-reaction signal at about 31 kDa,

Whereas S. cerevisiae produces the U. fabae protein PIG15p, the secretion of PIG15p could not be verified with this Western blot. Several possible conclusions can be drawn from the above results. The results will be discussed in chapter 5.2.7.

Figure 4-12 below displays a second Western blot for PIG15p, showing the occurrence of PIG15p protein in in vitro structures and haustorium of U. fabae:

Figure 4-12 Protein detection in in vitro structures by Western blot of using polyclonal antibodies against PIG15p. 10 µg protein were applied per lane. The lanes are labeled as follows: S: spore, 2h: infection structure after 2h (germ tube), 4h: infection structure after 4h (germ tube/ appressorium), 20h: infection structure after 20h (infection hypha), 24 h: infection structure after 24h (haustorial mother cell), H: haustoria.

The protein-extract from haustoria (lane H) and the protein-extract from non-germinated spores display an antibody cross-reaction signal at ca. 31 kDa. No signals are visible in the lanes with protein extract from germinated spores (in vitro structures).

Unfortunately, for PIG5p, it was not possible to produce a valid Western blot for heterologous protein expression and protein expression in haustoria. The antibody cross-reaction resulted in an indistinguishable smear over large sections of the respective lanes, making the identification of a clear signal impossible (results not shown). In addition, in the Western blots for PIG5p in vitro detection, no clear antibody cross-reaction signals were detected in the U. fabae spores, and no signals at all were detected in U. fabae in vitro infection structures (2h, 4h, 20h, 24h) using antibodies against PIG5p (results not shown).

The following table (Table 4-9) provides an overview and comparison between the theoretically calculated and the experimentally derived protein masses (by Western blot) for of PIG7p, PIG14p and PIG15p:

Table 4-9 Protein Mass and Occurrence of Studied in planta Induced Genes

4.9.2 Localization of PIG Products

In order to localize the gene products of the four remaining PIGs, slices of infected V. faba leaves were stained and examined using immuno-fluorescence microscopy. The primary polyclonal antibodies used in the protein-labeling process were the same as used for the Western blots described in chapter 4.9.1 above. The subsequent fluorescent staining with labeled secondary antibodies served to localize the gene products of the different PIGp in situ.

PIG7p (RTP1p) PIG14p PIG15p

[kDa] [kDa] [kDa]

Protein, calculated value 24.2 25.6 32.1

Protein without signal, calculated value (presumed mature protein)

22.1 23.7 29.6

His-tagged fusions-protein in E. coli 29 32 33

Transformed yeast (pDR195) pellet 25 / 32 80 / 90

29/32 30 / 32

Transformed yeast (pDR195) supernatant 100 120 Not detected

Transformed yeast (pDR195) supernatant deglycosylated

22 29 Not detected

Spore - - 31

2h infection structure Not detected Not detected Not detected

4h infection structure Not detected Not detected Not detected

20h infection structure Not detected Not detected Not detected

24h infection structure Not detected Not detected Not detected

Haustoria 23.5 / 25.0 29 31

The following series of photographs (Figure 4-13) shows the results of the immuno-fluorescence microscopy:

Figure 4-13 In situ localization of various PIGps in rust-infected bean leaves. The light-microscopy images of the plant structures are overlaid with fluorescence light-microscopy images of the indicated PIGp. (EHM): extrahaustorial membrane; (H): haustorium;

(N): plant nucleus; (P): plastids; (PC): plant cell. Staining and photography by Prof. Dr.

K. Mendgen and Dipl. Ing. (FH) H. Vahlenkamp.

As seen in the first photograph, PIG5p is located in the inside the haustoria. No staining of the extrahaustorial matrix or the host cells is visible.

As can be seen in the second and third photographs, the immuno-fluorescence staining clearly shows that PIG14p and PIG15p are both located in the extrahaustorial matrix at the fungal plant borderline. In both cases, the host cells are not stained.

As made visible in the fourth photograph, PIG7p (RTP1p) is located in the nucleus of the host V. faba. PIG7p can also be found in the extrahaustorial matrix und to a limited extend in the cytoplasm of the host, whereby none of the uninfected neighbor cells show a signal (results not shown).

The immuno-fluorescence staining provides strong evidence that PIG7p (RTP1p) is transferred from the rust haustoria into the nucleus of the host plant V. faba. This is the first time it has been shown that a plant pathogenic fungal protein is transferred to the nucleus of its host. The implications of this finding will be discussed in more detail in chapter 5.2.8.

5 Discussion