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J Gen Plant Pathol (2012) 78:255–259 DOI 10.1007/s10327-012-0389-3

FUNGAL DISEASES

FUNGAL DISEASES

Rice false smut pathogen, Ustilaginoidea virens, invades through small gap at the apex of a rice spikelet before heading

Taketo Ashizawa Mami Takahashi Michiyoshi Arai Tsutomu Arie

Received: 7 February 2012 / Accepted: 10 April 2012 / Published online: 13 June 2012 The Phytopathological Society of Japan and Springer 2012

Abstract Ustilaginoidea virens, the false smut pathogen of rice, produces false smut balls on spikelets after heading. To clarify how the fungus invades spikelets during the booting stage, we developed a fungal strain that expresses a green fluorescent protein gene and injected conidia from this strain into rice sheaths. Observations at 48 h post- inoculation showed many conidia were present on spikelet surfaces, and the conidia had germinated and the hyphae have gradually grown by 120 h post-inoculation. By 144 h, hyphae had invaded spikelets through their apices, via the small gap between the lemma and palea and had already reached all floral organs.

Keywords

fluorescent protein Conidia

Rice Spikelet Villosiclava Green

Rice false smut caused by Ustilaginoidea virens (Cooke) Takahashi (teleomorph Villosiclava virens) (Kepler et al. 2012; Tanaka et al. 2008; White et al. 2000) is a serious disease of agricultural rice. False smut balls, green with numerous chlamydospores, appear on rice spikelets, and contain ustiloxins that are poisonous to animals (Nakamura et al. 1992; Suwa 1915). Chlamydospores in paddy soils

T. Ashizawa (& ) M. Takahashi M. Arai Hokuriku Paddy Cropping System Project Team, Hokuriku Research Center, National Agricultural Research Center, National Agriculture and Food Research Organization, 1-2-1 Inada, Joetsu, Niigata 943-0193, Japan e-mail: toketa@affrc.go.jp

T. Arie Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan

are the primary source of infection (Ikegami 1963). The fungus then colonizes tissues of the growing points of til- lers during the vegetative stage of rice growth (Ikegami 1963) and is believed to be transferred to young panicles in leaf sheaths. As already reported by Ashizawa and Kataoka (2005), the fungus is present in panicles at the booting stage because nested-PCRs targeting the species-specific ITS region of ribosomal DNA have confirmed the presence of the fungus in whole panicles before rice heading. In addition, spray inoculation tests of rice plants in fields indicated that the infection did not occur after heading of plants (Fujita et al. 1989). After rice heading, we were able to clearly discriminate infected from uninfected spikelets on a panicle because infected spikelets contained the white fungus and finally formed smut balls (Ikegami 1961). Our hypothesis is that infection occurs in spikelets on panicles in the leaf sheath before heading. However, how the fungus invades spikelets during the booting (reproductive) stage has been unknown. A green fluorescent protein (GFP) from the jellyfish Aequorea victoria (Prasher et al. 1992; Shimomura et al. 1962) provided a means of labeling the fungus to observe its behavior in plant tissues under in vivo conditions under fluorescence microscopy. Visualization of the GFP-labeled fungus is effective and allows temporal analysis of inva- sion. A GFP-labeled strain of Ustilaginoidea virens was previously developed by Agrobacterium tumefaciens- mediated transformation (Zhang et al. 2006) or electro- poration (Tanaka et al. 2011). We have also developed a GFP-labeled strain of U. virens by polyethylene-glycol- mediated protoplast transformation and observed the initial infection of rice spikelets before heading. We used the false smut isolate U. virens U2003-1 (Ashizawa et al. 2010) to obtain protoplasts. Hyphal tips of U2003-1 were transferred into potato dextrose broth (PDB)

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a d e b f c
a
d
e
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f
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Fig. 1 Deposition, colonization, and invasion processes of a GFP-

indicates hyphae growing through the small gap (between lemma and

tagged Ustilaginoidea virens isolate, U2003-1, on surface of spikelets of rice variety Yumeaoba, viewed with fluorescence microscopy. Inoculated rice panicles were examined at 24–144 h post-inoculation (hpi) before heading and 9 and 11 days post-inoculation (dpi) after heading. a Numerous conidia on surface of spikelet, 48 hpi. Arrows indicate conidia that have not germinated. Upper right inset is a detail of spores on spikelet surface. White bar 20 lm, yellow bar 10 lm. b Hyphae (arrow) on surface of spikelet, 120 hpi. Bar 50 lm. c–g

palea) into the interior of spikelet. Bar 200 lm. White arrows indicate hyphae on inner surface of spikelet. d Hyphae on inner surface of spikelet. Bar 200 lm. e Hyphae (arrows) on anther. Bar 50 lm. f Hyphae on stigma (upper arrows) and anther (lower arrow). Bar 50 lm. g Hyphae (arrow) on lodicules. Bar 200 lm. h Hyphae covering anther, 9 dpi. Bar 50 lm. i Hyphae covering all floral organs, 11 dpi. Bar 100 lm. j Hyphal locations on rice floral organs based on observations in e–h: 1 upper anther (corresponding to e, h),

Hyphae at 144 hpi. c Hyphae at small ‘‘gap’’ at spikelet apex. Black

2

stigma (corresponding to f), 3 lower anther (corresponding to f),

arrows indicate mycelium on outer surface of spikelet. Blue arrow

4

lodicule (corresponding to g). Bar 1 mm

(Difco, Franklin Lakes, NJ, USA) and incubated at 28 C for 7 days with shaking at 120 rpm. Hyphae were then harvested from the liquid culture. The tubes were incubated on a horizontal shaker at 120 rpm for 4 days to obtain a conidial suspension (Ashizawa et al. 2011). Plasmid pMK412 (Watanabe et al. 2007) carrying the engineered GFP (EGFP) and the hygromycin B phospho- transferase gene was used for fungal transformation. The plasmid was propagated in Escherichia coli DH5a and

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purified using a Plasmid Mini Kit (Qiagen, Tokyo, Japan). One microgram per milliliter of the purified plasmid was linearized with 750 U XbaI in 50 lL Tris–EDTA (TE) at 37 C for 4 h and used to transform the fungus without inactivation of the enzyme. Hyphae of U2003-1, grown in PDB as described, were filtered and washed with OM buffer (1.2 M MgSO 4 , 10 mM sodium phosphate, pH 6.8). Washed hyphae were resuspended in 10 mL of filter-sterilized (pore diameter

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g i j h 1 2 3 4
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Fig. 1

continued

0.2 lm) OM buffer containing 30 mg/mL of lysing enzyme (Sigma, St. Louis, MO, USA) in a 50 mL conical tube. The tube was incubated at 28 C for 3.5 h by gentle horizontal shaking (60 rpm). The protoplast solution fil- tered through two-layered tissue paper was overlaid with 7.5 mL of ST buffer (1.2 M sorbitol, 10 mM Tris–HCl, pH 7.5) in a 50 mL conical tube. The tube was centrifuged at 40009g for 10 min. The protoplast layer (middle layer) was transferred to a new 50 mL conical tube and mixed with 15 mL of STC buffer (1.2 M sorbitol, 10 mM Tris– HCl, 20 mM CaCl 2 , pH 7.5). The protoplast solution was centrifuged at 20009g for 10 min, and suspended in 800 lL of STC buffer adjusted to a concentration of 5 9 10 7 protoplasts/mL. Eight hundred microliters of the protoplast suspension was mixed with 200 lL of XbaI-digested pMK412 in a 15 mL conical tube. One milliliter of polyethylene glycol (PEG) solution [60 % (w/v) PEG 4000, 10 mM Tris–HCl, 50 mM CaCl 2 ] was added twice to the mixture by droplet, kept for 18 min at 25 C and transferred to ice for 2 min. Forty milliliters of STC-50 buffer (1.2 M sorbitol, 10 mM Tris–HCl, 50 mM CaCl 2 , pH 8.0) was gently added and mixed, and centrifuged at 30009g for 10 min. The proto- plast solution was suspended in 10 mL of YG1/2SC

medium [0.5 % (w/v) yeast extract, 2.0 % (w/v) glucose, 0.6 M sorbitol, 25 mM CaCl 2 ], and incubated at 28 C for 3 h. The protoplast suspension was mixed with 300 mL of YG20S agar [0.5 % (w/v) yeast extract, 2.0 % (w/v) glu- cose, 20 % (w/v) sucrose, 1.5 % (w/v) agar] containing 100 lg/mL of hygromycin B. After incubation at 28 C for 7–10 days, emergent colonies were transferred to new YG20S agar containing 200 lg/mL of hygromycin B. Three GFP-transformants of U2003-1 were obtained. GFP- tagged transformants were selected using fluorescence microscopy (Leica, DN4000B, Tokyo, Japan) with UV filter set GFP Plant MZ FLIII and used for further study. Pathogenicity of the GFP-tagged U2003-1 to rice plants was previously tested and confirmed, and the fungus formed normal false smut balls (data not shown). We cultivated the main culms of plants of susceptible rice variety Yumeaoba for use in a rapid method to obtain conidial suspensions (Ashizawa et al. 2011). Briefly, five rice seeds were sown in 400 g of artificial soil (Honensu- baido1, Honen Agri, Niigata, Japan) in a pot (diameter 10.5 cm). Rice plants were allowed to grow only along their main culms, and emergent tillers were cut (Satake 1972). For inoculation of rice plants, eight dried barley seeds on which the isolate was growing were added to a

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a
a
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Fig. 2 Diagram of infection route from conidial deposition to floral organ invasion by the false smut fungus. a (1) Conidia (black spot) are deposited on outer spikelet surface at 48 h post-inoculation (hpi). Bar 1 mm. b (2) Conidia germinate, and hyphae (black lines) develop on outer spikelet surface until 120 hpi. Hyphal development near the spikelet apex (red circle) is crucial for further invasion. Bar 1 mm. c (3) Hyphae elongate from the small gap (yellow arrow) at the spikelet apex at 144 hpi onto the inner spikelet surface (black arrows). Bar 1 mm. Upper right inset is a detail of the small gap (yellow arrow). (4) Hyphae attach to the floral organs (red circle). Blue bar 200 lm

50 mL conical tube containing 25 mL of PDB and 2 % sucrose (w/v). The cultured broth was filtered through two layers of tissue paper to obtain a conidial suspension.

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At the booting stage of rice growth (when flag leaves have emerged), 2 mL of conidial suspension (5 9 10 5 conidia/ mL) were then injected onto the leaf sheaths of rice plants. Plants were transferred to an incubator at 16 C for 2 days, then moved to a moist chamber with 100 % relative humidity at 26 C for 5 days. After inoculation, plants were moved to a greenhouse controlled at 25 /20 C (day/ night). Nonheading, inoculated panicles within leaf sheaths were excised at 24, 48, 72, 96, 120, and 144 h post-inoc- ulation (hpi) to examine the infection process of U. virens. Whole, longitudinal, and cross sections of the panicles, and/or 0.5–1 mm thick sections of the panicle tissues were cut with a stainless steel razor blade, then observed with fluorescence microscopy. After heading, panicles were excised at 9 and 11 days post-inoculation (dpi). At 24 and 48 hpi, the inoculated panicles in the leaf sheaths were well wetted by the conidial suspension of GFP-tagged Ustilaginoidea virens isolate U2003-1, and GFP-tagged conidia were observed on the surfaces of spikelets (Fig. 1a). No germinated conidia were observed on any panicle surfaces or in the remaining conidial sus- pensions in leaf sheaths. At 72, 96, and 120 hpi, the conidia of GFP-tagged U. virens deposited on the spikelet surfaces were observed to have germinated and formed hyphae (Fig. 1b). At 24–120 hpi, no GFP-tagged U. virens were observed in the floral organs of the inner spikelets. At 144 hpi, hyphae had grown from the apices of spikelets through the small gap between the lemma and palea (Fig. 1c) and onto the inner surfaces of the lemma and palea (Fig. 1d). Furthermore, GFP-tagged U. virens had reached floral organs such as the anthers (Fig. 1e, f, j-1, j-3), stigmata (Fig. 1f, j-2), filaments and lodicules (Fig. 1g, j-4). At 24–144 hpi, the fungus was not observed in any inner tis- sues/cells of the spikelet organs, the panicle axis, primary and secondary branch pedicels, or the rudimentary glumes. After heading of rice plants, the floral organs were covered with hyphae by 9 dpi (Fig. 1h, j-1), and finally all the organs were completely covered with hyphae by 11 dpi (Fig. 1i). The hyphae in the spikelets appeared the same as the typically observed symptoms; the initial infected spikelets containing white hyphae. The route of spikelet invasion of U. virens is outlined in Fig. 2; (1) conidia land on the outer spikelet surface (Fig. 2a), (2) hyphae develop and grow on the outer spikelet surface (Fig. 2b), (3) then grow from spikelet apex onto the inner spikelet surface (Fig. 2c). (4) Finally, hyphae had grown onto the floral organs. We developed GFP-tagged U. virens to monitor fungal invasion of rice panicles. Consistently green-fluorescing U. virens was clearly distinguishable from the red or hyaline to yellow plant tissues using fluorescence microscopy. After inoculation and the 2-day, 16 C incubation, the conidia deposited on the spikelets and other panicle tissues

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had not germinated. Hyphae grew later, forming mycelia on the surfaces of spikelets to enable hyphal invasion into the floral organs in the spikelets. In fact, conidia in the inoculum suspension were still found within a leaf sheath with an unheaded panicle at 144 hpi, but germination was poor, and the hyphae were too short to reach floral organs (photo not shown). Interestingly, when cultured hyphae were injected into leaf sheaths, no infection occurred, nor did any false smut balls form. Thus, conidial deposition and subsequent hyphal development may be a crucial step for infection. The growth through the small gap also indicates that U. virens infects rice plants without pene- trating the cells and/or growing endophytically. The small gap invasion process may contribute to the random occurrence of false smut balls on panicles (Sonoda et al. 1988), because each random infection event results in a spikelet with well-developed hyphae attached near the small gap. During infection of the inner floral organs, lodicule invasion, in particular, may be important for fur- ther colonization by the hyphae. It is important for the fungus to stop the lodicules from swelling, which pushes apart the lemma, opening the spikelet (anthesis). Other air- borne microorganisms could then invade the spikelets. Interfering with lodicule swelling thus allows U. virens to prevent contamination from competing microorganisms, as well excessive drying and UV exposure. In fact, by 9–11 dpi, invading hyphae of U. virens had covered all the floral organs including the lodicules. Our findings should contribute to breeding rice varieties that are resistant to false smut, through selection of lines with much smaller or closed gaps and to the development of new fungicides to suppress invasion of spikelets at the booting stage.

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