Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

(1)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 1. Spatial Distribution of PI4P and PI(4,5)P

2

at Penetration Sites of the Powdery Mildew E. cichoracearum.

Leaves of Arabidopsis plants expressing biosensors were inoculated with Ec and viewed under a confocal microscope at 2 dpi.

Fungal structures and plant cell walls were stained with propidium iodide (PI). ha, haustorium; hn, haustorial neck, en, encasement.

Scale bars, 10 µm.

(A) and (B) Distribution of PI4P sensor mCIT-2xPH

^FAPP1

at the Ec-infection site. (A) The same inoculation site was focused sequentially at the peripheral surface (top), haustorial neck (middle) and haustorial body (bottom). (B) 3D view of maximum projection of Z-stacks.

(C) and (D) Distribution of PI(4,5)P

2

sensor mCIT-1xPH

^PLCδ1

at the Ec-infection site. (C) The same inoculation site was focused at the peripheral surface (top), haustorial neck (middle) and haustorial body (bottom). (D) 3D view of maximum projection of Z-stacks, showing that a layer of continuous mCIT-1xPH

^PLCδ1

signals appeared from the haustorial neck region to the EHM.

(Support Figure 1)

(2)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 2. Differential Distribution of Host Cellular Membrane Compartments in Association with the Haustorium of the Powdery Mildew E. cichoracearum.

Leaves of Arabidopsis plants expressing indicated probes were inoculated with Ec and viewed under a confocal microscope at 2

dpi. The probes included PI(4,5)P

2

sensors mCIT-1xPH

^PLCδ1

and mCIT-2xPH

^PLCδ1

, and fluorescent protein-tagged markers RPW8.2-

YFP for EHM, PMA-GFP for PM, Cyto-YFP for cytosol, Tono-GFP for tonoplast, and ER-GFP for ER. Fungal structures and plant

(3)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 3. Differential Distribution of Phosphoinositides to the Haustorial Periphery of Albugo candida.

Cotyledons of Arabidopsis plants expressing indicated biosensors were inoculated with zoospores of A. candida and viewed under a confocal microscope at 2 dpi. ha, haustorium. Scale bars, 5 µm.

(A) Representative images of PI3P biosensor mCIT-2xFYVE

^HRS

. (B) Representative images of PI4P biosensor mCIT-2xPH

^FAPP1

.

(C) Representative images of PI(4,5)P

2

biosensors mCIT-2xPH

^PLCδ1

and mCIT-1xPH

^PLCδ1

.

(Support Figure 2)

(4)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 4. Increased Production of PI(4,5)P2 in Ec-colonized Leaf Epidermal Cells.

Representative confocal images of Ec-infected epidermal cells of Col-0 plants expressing UBQ10:mCIT-1xPHPLCδ1 as a biosensor for PI(4,5)P2 or UBQ10:EYFP for free YFP under the same promoter. Images were taken at 24 hpi at the same confocal setting and viewed with maximum projection of Z-stacks. Infected cells are indicated by arrowheads. Induced accumulation of UBQ10:mCIT- 1xPHPLCδ1 was observed in 47 of 79 Ec-colonized cells, whereas no induction of pUBQ10:YFP was observed in ~80 Ec-colonized cells.

app, appressorium; ha, haustorium. Scale bars, 50 µm.

(Support Figure 4)

(5)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 5. Expression Profiling of Arabidopsis PI4P 5-kinase Genes and Characterization of T-DNA Insertion Mutants.

(A) Tissue-specific expression of eleven Arabidopsis PI4P 5-kinase genes. Public affymetrix DNA microarray data sets for Arabidopsis were mined to generate a heat map using Genevestigator (https://www.genevestigator.com). Predicted expression levels are indicated in the left-top corner inset.

(B) RT-PCR analysis of Arabidopsis PI4P 5-kinases and defense-related genes in Arabidopsis leaves in response to Ec inoculation.

Leaves of 4-week-old Col-0 plants without or with Ec inoculation were harvested for RNA extraction at indicated time points. The level of elF4A1 was used as an internal control for normalization. PCR products were amplified from 28 cycles.

(C) Genomic organization and location of T-DNA insertion sites of leaf expressed PI4P 5-kinase genes in Arabidopsis. White boxes represent coding DNA sequences (CDS), black lines represent introns, gray bars correspond to untranslated regions (UTR), and red triangles indicate T-DNA locations. Sizes of CDS, TUR and introns were drawn with Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn) according to the TAIR 10 Gene Models.

(D) Development of Ec on leaves of single or double mutants. Representative images of Arabidopsis leaves of the indicated genotypes show colony morphology at 7 dpi. Fungi were stained with aniline blue, and arrowheads indicate the conidiospores that cause initial infection. Scale bars, 100 µm.

(Support Figure 5)

(6)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 6. Growth Phenotypes of the Arabidopsis Knockout Mutants and Complemented Mutant Plants of PI4P 5- kinase Genes.

Plants with the indicated genotypes were selected by PCR, grown on soil, including Col-0 wild type, pip5k1 pip5k2 double mutant, pip5k1 pip5k2 pip5k5 triple mutant, pip5k1 pip5k2 pip5k8 triple mutant, pip5k1 pip5k2 pip5k5 pip5k8 quadruple mutant, and the pip5k1 pip5k2 double mutants ectopically expressing PIP5K1-YFP or PIP5K2-YFP under their native promoter. Scale bar, 5 cm.

(A) 4-week old plants.

(B) 6-week-old plants.

(Support Figure 5)

(7)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 7. Pathogenicity Assays on Col-0 and pip5k Double, Triple and Quadruple Mutant Plants against the White Rust Albogo candida.

Macroscopic infection phenotypes of indicated genotypes at 15 dpi with A. candida isolate Aecm1. Leaves were inoculated with droplets of zoospore suspension on adaxial leaf surface. Inoculation sites were indicated by dotted circles. Infected leaves were photographed at 15 dpi.

(Support Figure 5)

(8)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 8. Impaired Cellular Levels of PI(4,5)P

2

in pip5k1 pip5k2 Mutants.

(A) and (B) Cellular distribution of PI(4,5)P

2

in leaf epidermal tissue of Col-0 and pip5k1 pip5k2 plants expressing mCIT-1xPH

^PLCδ1

. (A) The same sites were viewed on the peripheral surface (left panel, single image), inside the cell (middle panel, single image) or 3D with Z-stack (right panel, maximum projection) of the leaf epidermis. Scale bars, 20 µm. (B) Relative fluorescence intensity of mCIT-1xPH

^PLCδ1

at the PM. Data are means ± SD, n = 30. **P < 0.01, student t-test.

(C) Expression profiling of mCIT-1xPH

^PLCδ1

transgene in pip5k1 pip5k2 and Col-0. RT-PCR of mCIT-1xPH

^PLCδ1

was performed on total RNA extracted from 4 weeks Arabidopsis leaf tissues. The primer pairs are located to the mCITRINE region. The transcript level of elF4A1 was used as an internal control to normalize amount of cDNA template.

(D) Impaired targeting of mCIT-1xPH

^PLCδ1

to the EHM in the pip5k1 pip5k2 mutant. Representative confocal images were taken at 24 hpi at the same confocal setting. Scale bars, 10 µm. Relative fluorescence intensity of mCIT-1xPH

^PLCδ1

was measured at the EHM. Data are means ± SD, n = 30. **P < 0.01, student t-test.

(E) Increased PI(4,5)P

2

signals in leaf epidermal cells harbouring a haustorium of Ec. Representative confocal images of Ec-infected epidermal cells of Col-0 and pip5k1 pip5k2 plants expressing UBQ10:mCIT-1xPH

^PLCδ1

. Images were taken at 24 hpi at the same confocal setting and viewed with maximum projection of Z-stacks. Infected cells are indicated by arrowheads. Scale bars, 50 µm.

EHM, extrahaustorial membrane; ha, haustorium.

(Support Figure 5)

(9)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 9. Cellular Localization of PIP5K1-YFP and PIP5K2-YFP in Arabidopsis.

(A) and (B) Expression and cellular location of PIP5K1-YFP and PIP5K2-YFP in Arabidopsis leaf tissues. True leaves of Arabidopsis plants expressing pPIP5K1:PIP5K1-YFP (A) and pPIP5K2:PIP5K2-YFP (B) were stained with PI and examined under confocal microscope before plasmolysis (upper panel) or after plasmolysis (lower panel).

(C) and (D) Both PIP5K1-YFP and PIP5K2-YFP focally accumulated at Ec penetration sites, but were absent from the EHM.

Arabidopsis plants expressing pPIP5K1:PIP5K1-YFP (C) and pPIP5K2:PIP5K2-YFP (D) were inoculated with Ec. The infection sites (arrowheads) were examined under confocal microscope with PI staining at 24 hpi. The same sites were viewed on the peripheral surface (upper panel) or inside the cell (lower panel) of the leaf epidermis.

ha, haustorium; app, appressorium. Scale bars, 20 µm.

(Support Figure 5)

(10)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 10. Expression levels of PR1, PDF1.2a, and FRK1 genes in Col-0 Wild Type and pip5k1 pip5k2 at 2, 5 and 7 Days after Inoculation with E. cichoracearum.

Normalized counts were plotted. Data are means ± SD, n = 3. Different letters indicate statistically significant differences determined by one-way ANOVA with Tukey’s HSD (p < 0.01).

(Support Figure 6)

(11)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 11. Cellular Dynamics of MLO2 and Reorganization of Actin Filaments in pip5k1 pip5k2 Mutant.

(12)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

(B) Apoplastic depositions of MLO2-GFP at the Ec-penetration sites. Ec-inoculated leaves of Col-0 expressing MLO2-GFP were stained with PI at 14 dpi, and images were captured after plasmolysis. Arrowheads indicate the boundary of retracting PM from the cell walls.

(C) Dynamic responses of MLO2-GFP on pip5k1 pip5k2 leaves at ~14 h post Ec inoculation. Left panel, an Ec-infection site associated with failed penetration attempt; right panel, an Ec-infection site associated with a successful penetration event. GFP signals were captured with confocal microscopy at identical setting for all images.

(D) Expression profiling of MLO2-GFP transgene in pip5k1 pip5k2 and Col-0 wildtype. RT-PCR of MLO2-GFP transgene was performed on total RNA extracted from 4 weeks Arabidopsis leaf tissues of indicated genotypes. The primer pairs are targeted to the GFP region. The transcript level of elF4A1 was used as an internal control to normalize amount of cDNA template.

(E) Altered organization of actin filaments in leaf epidermal cells of pip5k1 pip5k2 mutant. Representative confocal images of leaf epidermal cells of Col-0 wild type and pip5k1 pip5k2 plants expressing GFP-ABD2-GFP. Images were taken at the same confocal setting with a maximum-projection of Z-stacks.

Asterisks indicate the contact sites underneath conidia. Arrowheads indicate the penetration sites. app, appressorium; pp, penetration peg. Scale bars, 20 µm.

(Support Figure 7)

(13)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Figure 12. Loss of PIP5K1 and PIP5K2 Functions Prevented Disease Development of C. higginsianum Infection on Detached Arabidopsis Leaves.

Detached leaves of Col-0 wild type and pip5k1 pip5k2 plants were inoculated with droplets of Ch conidiospore suspension.

Inoculated leaves were incubated in a moist chamber at 20 °C. Images were taken during an infection time course. At 6 dpi, both

adaxial and abaxial surfaces of inoculated leaves of pip5k1 pip5k2 mutant were viewed, showing the restricted lesions within the

inoculated area.

(14)

Supplemental Data. Qin et al. (2020). Plant Cell 10.1105/tpc.19.00970

Supplemental Table 1. Frequency at Which Biosensors Were Observed at the Extrahaustorial Membrane of Powdery Mildew E. cichoracearum.

PIPs Biosensors Positive/total* Frequency

PI4P mCIT-2xPH

^FAPP1

(P21Y) 0/77 0%

mCIT-1xPH

^FAPP1

(P5Y) 0/38 0%

mCIT-P4M

^SiDM

(P4M) 0/33 0%

PI(4,5)P

2

mCIT-1xPH

^PLCδ1

(P14Y) 111/111 100%

mCIT-2xPH

^PLCδ1

(P24Y) 37/37 100%

mCIT-1xTUBBY-C (P15Y) 42/42 100%

*data means the number of haustoria which had biosensor signals at their extrahaustorial membrane in the total number of

haustoria observed from at least 5 plants (one leaf/plant) per plant line at 48 hpi.

(15)