Imp1^GFP membrane localization is dependent on autophagy induction during growth in rice cells.

(A) Imp1GFP localization on the IH membrane and vacuole morphology during growth in rice cells is dependent on autophagy induction. Leaf sheaths infected with the Δimp1 IMP1GFP complementation strain expressing Imp1GFP were treated with 5 mM 3-methyladenine (3-MA) at 36 hpi and viewed at 44 hpi. Scale bars = 10 μm, arrows indicate appressoria on the leaf sheath surface. NT = no treatment. Proportion of infected rice cells represented by these images are shown in S2 Table. (B) Model showing the TOR-dependent role of Imp1 and autophagy in facilitating the lifespan of the BIC and EIHM, collectively referred to as the biotrophic interface. Although Imp1 is required for autophagy induction and is involved in V-ATPase function and the fusion of autophagosomes and endosomes to vacuoles, pharmacological evidence situates the role of Imp1 in biotrophic interface maintenance and effector deployment upstream of V-ATPase activity and downstream of phagophore initiation. Also, because 1) the number of autophagosomes are decreased in Δimp1; 2) endosomes can contribute membranes to phagosomes, and 3) because AM elevates early autophagy by increasing autophagosome number; we suggest that Imp1 acts by facilitating the contribution of endosomal membranes, originating at the plasma membrane, to phagophore expansion. Furthermore, 3-MA inhibition of autophagy induction prevented Imp1 localization on IH membranes, suggesting Imp1 might facilitate membrane recycling through the wider vesicular network or—because ConA and BafA1 treatment did not prevent Imp1 localizing on IH membranes—via a vacuole-independent route. Loss of Imp1 would reduce the efficiency of this membrane recycling process, leading to stochastic senescence of the biotrophic membrane over time. We thus propose that in response to autophagy initiation, Imp1 has two distinct roles: in vacuole function and late stage autophagy to optimize biotrophic growth, and in facilitating biotrophic interface longevity by mediating membrane sourcing during phagophore expansion and autophagosome formation.</p

<i>IMP1</i> mediates endocytic and autophagic vesicle dynamics.

(A) FM4-64 staining shows endocytic vesicles targeting vacuoles in Δimp1 and WT. After 5h, FM4-64 is not distributed throughout the full vesicular network in Δimp1 mycelia. Scale bar = 2 μm. (B) Monodansylcadaverine (MDC) staining revealed both reduced numbers of autophagic vacuoles in Δimp1 compared to WT, and reduced overlap with FM4-64 stained compartments. Mycelia from WT and Δimp1 were incubated in GMM for 16 h then stained with 1 μg per ml FM4-64 and 40 μM MDC for 5 h in water. Scale bar = 10 μm. (C) IMP1 is required for endosomal and autophagosomal membrane vesicle trafficking to acidified autophagic vacuoles, and might also be required for supplying endosomal membranes for phagophore expansion.</p

<i>IMP1</i> acts downstream of TOR kinase in the TOR-autophagy signaling branch.

(A) Spore suspensions of WT, Δimp1 and Δimp1 IMP1GFP strains were either not treated (NT) or treated with rapamycin to a final concentration of 200 nM and applied to non-inductive hydrophilic glass slides. Bars are the mean percentage of the number of appressoria formed from 50 germinating spores, replicated on three different slides, by 24 hpi. Bars with different letters indicate significant difference (α ≤ 0.05, LSD). Error bars are s.d. (B) Western blot showing the phosphorylation status of the direct TOR kinase target Sch9 in the indicated strains following treatment with 1 μM rapamycin (Rap) for 8h. Strains were grown in complete media (CM). NT = no treatment. RI = relative intensity calculated by normalizing Sch9 phosphorylation levels determined using anti-p-p70 S6 kinase antibody against tubulin α levels determined by anti-tubulin α antibody. Red lines mark lane boundaries used for densitometry. When the ends of neighbouring bands fused during gel running and made lane demarcation difficult, the boundary was placed between adjacent band tails. (C) Quantitative real-time PCR (qPCR) analysis of TOR-regulated genes in the indicated strains following 16 h growth in CM. NT = no treatment; Rap = 1 μM rapamycin treatment. Data represent mean values ± s.d. from two biological replicates with three technical replicates each (Student’s t test **p ≤ 0.001, ***p ≤ 0.0001), normalized against the expression of TUB2 encoding β-tubulin. (D) IMP1 is not required for the utilization of alternative nitrogen sources. Left, qPCR analysis shows IMP1 is not required for the rapamycin-induced derepression of the nitrate reductase structural gene (NIA1). NT = no treatment; Rap = 1 μM rapamycin treatment. Data represent mean values ± s.d. from two biological replicates each with three technical replicates (Student’s t test **p ≤ 0.001, ***p ≤ 0.0001), normalized against the expression of TUB2 encoding β-tubulin. Right, IMP1 is not required for utilizing nitrate as an alternative nitrogen source. Strains were grown for 12 days on MM with 1% (w/v) glucose (Glc) and 10 mM nitrate (NO3-) as the sole carbon and nitrogen source, respectively. (E) Growth of WT, Δimp1 and the Δimp1::IMP1GFP complementation strain on complete media (CM) and 2% water agar (WA) after 10 days. For clarity, black lines indicate colony diameters. (D,E) Representative images from three experiments are shown. (F) Model showing the proposed relationship between rapamycin, TOR signaling and Imp1. Alt N is alternative nitrogen source utilization.</p

Imp1^GFP localizes to the vacuole.

(A-D) Imp1GFP localizes to vacuoles in vegetative mycelia after axenic growth for 16 h in liquid MM with glucose as the sole carbon source (GMM). (B) Mycelia were stained with mitotracker for 30 min before subjected to scanning laser confocal microscopy. (A-B) Scale bar = 10 μm. (C) Imp1GFP localized to vacuoles associated with vesicles (arrows). Scale bar = 2.5 μm. (D) Imp1GFP co-localized with the specific vacuolar stain FM4-64. Scale bar = 10 μm. (E) Leaf sheath infection assays showed that after penetration into epidermal cells, Imp1GFP localized to a single vacuole and the IH plasma membrane at 28 hpi (left). By 44 hpi (right) Imp1GFP localized to many internal compartments, the IH plasma membrane, and vacuoles at the tips of IH emerging into cells adjacent to primary infected cells (thin arrow in zoom box). Large arrows indicate appressoria on the leaf sheath surfaces. Scale bar = 10 μm. (F) Western blot analysis of Imp1GFP using anti-GFP monoclonal antibodies to probe proteins extracted from vegetative mycelia (left), and from inoculated (+) and uninoculated (-) rice leaf sheaths (right). α-tubulin was used as the loading control. To explore whether growth conditions affect Imp1GFP processing or modification, vegetative mycelia were grown in MM under normal 1% w/v glucose (Glc) sufficient conditions (+), or under glucose restrictive (0.025% w/v) conditions (-), with or without 1 μM rapamycin (Rap).</p

<i>IMP1</i> confers rapamycin sensitivity and is required for rice blast disease.

(A) Agrobacterium tumefaciens-mediated transformation generated a rapamycin resistant strain, AT2, resulting from a T-DNA insertion event at MGG_08120 encoding an integral membrane protein (Imp1). LB and RB are the known left flank and right flank T-DNA sequences, respectively. (B) Targeted deletion of IMP1 in the wild type (WT) strain Guy11 recapitulated AT2 by conferring rapamycin resistance to Δimp1 strains. Δimp1 IMP1GFP is the Δimp1 mutant strain complemented with the IMP1 gene fused to the GFP-encoding cassette. Strains were grown on minimal media (MM) containing 1% (w/v) glucose and 10 mM NH4+ as the sole carbon and nitrogen source, respectively, and on MM supplemented with 10 μM rapamycin (Rap), for 12 days. (C) The Δimp1 mutant strain was non-pathogenic on seedlings of the susceptible rice cultivar CO-39 compared to WT and the Δimp1 IMP1GFP complementation strain. (D) Loss of IMP1 marginally reduced appressorium formation rates on rice leaf surfaces compared to WT by 36 hpi. Values are the average percentage of appressoria formed by 50 germinating spores of each strain per rice cuticle, repeated in triplicate. (E) The penetration of Δimp1 appressoria through the rice cuticle and into underlying epidermal cells was reduced but not abolished compared to WT and the Δimp1 IMP1GFP complementation strain. Bars are the average percentage of penetration pegs developed at 36 hpi by 50 appressoria of each strain per rice cuticle, repeated in triplicate. (F) At 44 hpi, the Δimp1 mutant strain was impaired in cell-to-cell movement compared to WT and the Δimp1 IMP1GFP complementation strain. Bars are the average of 50 primary infected rice cells from which invasive hyphae (IH) were shown emerging into adjacent rice cells. Experiments were repeated in triplicate. (D-F) Error bars are s.d. Bars with different letters are significantly different (α ≤ 0.05, Least significant difference (LSD)).</p

<i>IMP1</i> maintains biotrophic interface integrity.

WT (A) and Δimp1 (B) strains expressing the fluorescently labeled apoplastic effector Bas4GFP and the fluorescent BIC-accumulating cytoplasmic effector Pwl2mCherry:NLS at 32 hpi. White arrows indicate appressoria on the leaf sheath surface and red arrow indicates the faint enrichment of Pwl2mCherry:NLS in an adjacent rice nucleus. Arrowheads highlight Bas4GFP in the apoplast. Scale bars = 10 μm. Percentages are mean values +/- s.d. of each representative image obtained from observing 100 infected rice cells per strain, repeated in triplicate.</p

<i>IMP1</i> is required for biotrophic interface longevity.

WT (A) and Δimp1 (B) strains expressing the fluorescently labeled apoplastic effector Bas4GFP and the fluorescent BIC-accumulating cytoplasmic effector Pwl2mCherry:NLS at 44 hpi. Stars indicate emerging IH in adjacent cells. Arrows indicate appressoria on the leaf sheath surface. Scale bars = 10 μm. Percentages are mean values +/- s.d. of each representative image obtained from observing 100 infected rice cells per strain, repeated in triplicate. (C) Representative images of Δimp1 expressing Bas4GFP and Pwl2mCherry:NLS and infecting rice cells at 28 hpi. Small white arrows indicate appressoria on the leaf sheath surface. Large red arrow indicates accumulation of Pwl2mCherry:NLS in an adjacent rice nucleus. Scale bars = 10 μm.</p

<i>IMP1</i> is required for V-ATPase assembly.

(A) Subcellular localization of Vma2GFP, the fluorescently labeled V1 domain subunit B, was misregulated in Δimp1 vegetative mycelium compared to WT. GMM = glucose minimal media. Scale bars = 10 μm. (B) Model of the relationship between TOR, Imp1 and V-ATPase assembly and activity. (C) Vma2GFP subcellular localization was examined in WT and Δimp1 strains during in planta growth at 44 hpi. Scale bars = 10 μm. White arrows indicate appressoria on the leaf sheath surface. (D) Immunoblot assessment of Vma2GFP integrity using anti-GFP monoclonal antibodies. Proteins were extracted from vegetative mycelia (left) and infected rice leaf sheaths (right). α-tubulin was used as the loading control. Vegetative mycelia of the VMA2GFP strain were grown in GMM for 16 hr. Infected leaf sheath were sampled at 44 hpi.</p

<i>IMP1</i> is required for organelle acidification in response to TOR signaling.

(A) Staining of acidified compartments (including vacuoles) in vegetative mycelia with 1 μg/mL quinacrine after growth in the indicated treatments for 3 h. Rap = 1 μM rapamycin. AM = 1 μM amiodarone hydrochloride, a TOR-independent autophagy stimulator. GMM = glucose minimal media. Scale bar = 5 μm. Red dashed circles indicate examples of quinacrine-stained acidified vacuoles. Blue dashed circles indicate examples of unstained vacuoles. (B) Bars are the average number of quinacrine-positive vacuoles per 10 μm length of mycelia that were counted in 30 mycelia fragments longer than 100 μm, repeated in triplicate. *** indicates p value Students t-test. Error bars are s.d. Mycelial length was measured by ImageJ. Contrast was adjusted to best distinguish vacuole boundaries for counting. (C) Imp1 acts downstream of TOR in the autophagy signaling branch and is required for vacuole acidification after autophagy induction by rapamycin treatment or nutrient starvation.</p

V-ATPase function is not required for biotrophic interfacial membrane integrity.

Leaf sheaths infected with the indicated strains were treated with (A) 10μM concanamycin A (ConA) or (B) 1 μM bafilomycin A1 (BafA1) at 36 hpi and viewed at 44 hpi. (A,B) Arrows indicate appressoria on the leaf surface. Scale bars = 10 μm. NT = no treatment. Proportion of infected rice cells represented by these images are shown in S1 Table. (C) BafA1 and ConA treatments suggest V-ATPase and vacuole function is required for cell-to-cell movement but not for maintaining biotrophic interfaces, which therefore must be dependent on an earlier stage of autophagy.</p