Investigation of rice blast development in susceptible andresistant rice cultivars using a gfp-expressing Magnaportheoryzae isolate

L. Campos-Sorianoa, G. Val�ebc, E. Lupottoc and B. San Segundoa*aDepartment of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Edifici CRAG, Campus

UAB, Bellaterra, Cerdanyola del Vall�es, 08193, Barcelona, Spain; bGenomics Research Centre, Agricultural Research Council (CRA), Via

S. Protaso, Fiorenzuola D’Arda, Piacenza; and cRice Research Unit, Agricultural Research Council (CRA), s.s.11 to Turin km 2.5, 13100,

Vercelli, Italy

In this study, an isolate of Magnaporthe oryzae expressing the green fluorescent protein gene (gfp) was used to monitor

early events in the interaction of M. oryzae with resistant rice cultivars harbouring a blast resistance (R) gene. In the

resistant cultivars Saber and TeQing (Pib gene), M. oryzae spores germinated normally on the leaf surface but pro-

duced morphologically abnormal germ tubes. Germling growth and development were markedly and adversely affected

in leaves of these resistant cultivars. Penetration of host cells was never seen, supporting the idea that disruption of

germling development on the leaf surface might be one of the resistance mechanisms associated with Pib function.

Thus, this particular R gene appeared to function in the absence of host penetration by the fungal pathogen. Confocal

laser scanning microscopy of M. oryzae-infected susceptible rice cultivars showed the dimorphic growth pattern that is

typically observed during the biotrophic and necrotrophic stages of leaf colonization in susceptible cultivars. The suit-

ability of the gfp-expressing M. oryzae isolate for further research on R-gene function and identification of resistant

genotypes in rice germplasm collections is discussed.

Keywords: blast, confocal laser scanning microscopy, green fluorescent protein, Oryza sativa, resistance genes

Introduction

Magnaporthe oryzae is the causal agent of rice blast, themost economically important fungal disease of cultivatedrice (Oryza sativa) worldwide (Ou, 1985). The blast fun-gus infects both temperate and tropical rice grown underdifferent ecosystems (i.e. upland, lowland, irrigated andrainfed) and causes more damage in areas where high-input agricultural systems are adopted. Based on multi-locus genealogy and mating experiments, M. oryzae wasdefined as a new species, separate from Magnaporthe gri-sea (Couch & Kohn, 2002). Thus, the Magnaporthecomplex comprises two distinct clades, one that includesisolates infecting Digitaria (crabgrass), referred to asM. grisea isolates, and a second one that includes isolatespathogenic on rice, millet and other grasses, referred toas M. oryzae.Magnaporthe oryzae has been described as a hemibio-

trophic pathogen which first establishes a biotrophicinteraction with the rice plant and later switches to adestructive necrotrophic lifestyle. Foliar infection is initi-ated by attachment of a three-celled spore of M. oryzae

to the rice leaf cuticle. A polarized germ tube emergesfrom the spore and grows on the leaf surface, before dif-ferentiating into the dome-shaped appressorium. Onceformed, the appressorium matures and generates enor-mous cellular turgor that is sufficient to rupture the plantcuticle (Wilson & Talbot, 2009). The appressoriumforms a specialized hypha, the penetration peg, which inthe lumen of epidermal cells expands to become a fila-mentous primary hypha. The infection proceeds with thedifferentiation of primary hyphae into bulbous invasivehyphae that fill the invaded cells (Kankanala et al., 2007;Wilson & Talbot, 2009). Several days after infection,blast lesions appear, in which the fungus sporulates pro-fusely, thus allowing the disease to spread rapidly toadjacent rice plants. Very recently, the fungus M. oryzaewas identified as the most damaging plant pathogenicfungus (Dean et al., 2012). At present, blast disease con-trol worldwide relies on the combination of chemicalfungicides and integrated cultural practices.In recent years, plant breeding has achieved significant

progress towards the enhancement of blast resistancewith the identification of a broad array of blast resistance(R) genes. To date, more than 85 blast R genes have beenidentified (Ballini et al., 2008) and some of them havealso been molecularly characterized: Pib (Wang et al.,1999), Pita (Bryan et al., 2000), Pi9 (Qu et al., 2006),Pi2/Pizt (Zhou et al., 2006), Pid2 (Chen et al., 2006),Pi36 (Liu et al., 2007), Pi37 (Lin et al., 2007), Pik-m

*E-mail: blanca.sansegundo@cragenomica.es

Published online 13 December 2012

1030 ª 2012 British Society for Plant Pathology

Plant Pathology (2013) 62, 1030–1037 Doi: 10.1111/ppa.12023

(Ashikawa et al., 2008), Pi5 (Lee et al., 2009b), Pit (Hay-ashi & Yoshida, 2009), Pid3 (Shang et al., 2009) andPi21 (Fukuoka et al., 2009). Several of these blast resis-tance genes have demonstrated their ability to conferresistance to various blast pathotypes, and are beingeffectively used in breeding programmes to increase blastresistance in rice. However, none of the blast resistancegenes so far identified appears to confer resistance againstall isolates of M. oryzae. Because of the genetic variabil-ity of M. oryzae and race changes in blast populations,resistant cultivars with single-gene resistance have beenshown to lose effectiveness after a few years (Lee et al.,2009a). Durable resistance in breeding programmesrequires gene pyramiding strategies for the simultaneousexpression of more than one R gene in the same cultivar.To this end, molecular markers tightly linked to majorblast resistance genes are being used for marker-assistedselection (MAS) in rice (Fjellstrom et al., 2004). Recently,molecular markers for a panel of 13 blast resistance genesfrom 25 rice donor parental genotypes were reported(Tacconi et al., 2010), this information being useful toaddress pyramiding strategies in rice breeding. However,R-gene-mediated mechanisms and processes involved inblast disease resistance require further investigation.The usefulness of fungal isolates expressing the gfp

(green fluorescent protein (GFP)) gene to monitor fungalinfection of host plants is well demonstrated. Various fungihave been transformed with the gfp gene, including Ustil-ago maydis,M. oryzae, Fusarium oxysporum and Scleroti-nia sclerotiorum, among others (Sesma & Osbourn, 2004;Campos-Soriano & San Segundo, 2009; de Silva et al.,2009; Zvirin et al., 2010). This approach offers the possi-bility of monitoring in planta the invasion process andgrowth of the fungus in living tissues, using fluorescencemicroscopy without any further manipulation.In this work, a gfp-expressing isolate of M. oryzae

(PR9) and confocal laser scanning microscopy (CLSM)were used to visualize the early stages of fungal growthand invasion of susceptible rice cultivars. Moreover,CLSM allowed the in vivo imaging of M. oryzae growthin leaves of rice genotypes carrying blast resistance genes.Spore germination and the fate of germlings were exam-ined in more detail in resistant rice genotypes.

Materials and methods

Plant and fungal materials

A series of resistant and susceptible rice genotypes was chosenfor investigation. They were: Vialone Nano and Maratelli, two

Italian cultivars characterized as highly susceptible to blast;

Senia, a susceptible Spanish cultivar; Katy, Saber and Kanto 51,

three American cultivars resistant to blast; and TeQing, a highlyresistant Chinese cultivar. The resistant genotypes were charac-

terized by the presence of resistance genes: Pik in Kanto 51,

Pita2 in Katy, and Pib in Saber and TeQing (Tacconi et al.,2010). Rice genotypes TeQing, Saber and Katy were obtainedfrom Dr Harold Bockelman (USDA, Agricultural Research Ser-

vice, National Small Grain Research Facility, National Small

Grains Collection, USA), whereas Kanto 51 was provided by Dr

Kazutoshi Okuno (National Institute of Agrobiological Sciences,

Japan). Pure seed stocks came from the rice germplasm seedbank of the Consiglio per la Ricerca e la Sperimentazione in

Agricoltura (CRA-Rice Research Unit, Vercelli, Italy).

The gfp-expressing M. oryzae isolate PR9 described by Cam-

pos-Soriano & San Segundo (2009) was used in this study. Thisisolate expresses the sgfp gene under the control of the P. tritici-repentis ToxA gene promoter (Sesma & Osbourn, 2004). The

fungus was grown on oat medium (Difco) for 2 weeks. Sporeswere collected by adding sterile water to the surface of the

mycelium. After filtration through sterile Miracloth (Calbio-

chem), spores were adjusted to the appropriate concentration

with sterile water using a B€urker counting chamber.

Infection assays

Infection experiments with the gfp-expressing M. oryzae isolate

(gfp-PR9) were carried out using the detached leaf assay as previ-

ously described (Coca et al., 2004). Briefly, the second leaves ofrice plantlets at the three-leaf stage were placed into agar plates

(1% w/v in water) containing 2 mg kinetin L�1. Whatman filter

paper discs saturated with a M. oryzae spore suspension at the

appropriate concentration were placed onto the adaxial leaf sur-face. The inoculated leaves were maintained in the dark in a

chamber under high humidity conditions for 48 h, after which

the filter paper discs were removed. Leaves were maintained at

28°C and 90% relative humidity under a 16-/8-h light/dark pho-toperiod for the required period of time. Microscopic examina-

tions of the infected tissue were carried out with three different

spore concentrations (104, 105 and 106 spores mL�1). Disease

symptoms were evaluated 3 and 6 days post-inoculation (dpi).Four independent experiments were carried out for each rice cul-

tivar. In each experiment, at least three leaves were inoculated

with each spore concentration (five inoculation points per leaf).Lesion areas were measured by image analysis software ASSESS v.

2.0 for plant disease quantification (Lamari, 2008).

Microscopy

Rice leaves were subjected to CLSM analysis at 3, 6, 30, 54, 60

and 72 h after inoculation with M. oryzae spores using anOlympus Fluoview FV1000. For visualization of GFP fluores-

cence, the excitation wavelength was 488 nm and the emission

window was set at 500–550 nm. For visualization of chloro-phyll autofluorescence, the same excitation wavelength was used

and the emission window was set at 600–700 nm. Two indepen-

dent experiments, with 10–20 leaves (five inoculation points per

leaf) in each, were carried out.

Results

Macroscopic evaluation of disease severity

During a plant–pathogen interaction, a complex set ofreactions must occur in the two partners, host andpathogen, that ultimately influences the outcome of theirinteraction. The particular aim of this study was to char-acterize early events occurring during the rice–M. oryzaeinteraction. To this end, several rice cultivars were selectedfor which phenotypes of resistance or susceptibility toblast infection had been previously demonstrated underfield conditions (Katsantonis et al., 2007; Tacconi et al.,

Plant Pathology (2013) 62, 1030–1037

M. oryzae development in resistant and susceptible rice 1031

2010; Faivre-Rampant et al., 2011). Among the resistantcultivars chosen were TeQing, Saber, Katy and Kanto 51,which each carry a Pi gene in their genome, namely Pib(Saber and TeQing), Pik (Kanto 51) or Pita2 (Katy). BothTeQing and Saber (Pib gene) showed a high level of resis-tance to various M. oryzae isolates at different plantdevelopmental stages under natural field conditions (Tac-coni et al., 2010). Kanto 51 and Katy (Pik and Pita2

genes, respectively) exhibited resistance to blast infectionat the tillering and stem elongation stages, but a low levelof infection at the heading and milk stages of plant growth(Tacconi et al., 2010). The susceptible cultivars assayed inthe present study showed moderate (Senia) to high (Mara-telli, Vialone Nano) sensitivity to blast.For blast disease assays, rice leaves were locally inocu-

lated with increasing doses of spores from the gfp-express-ing M. oryzae isolate to give rise to a macroscopicallyvisible lesion. As previously reported (Campos-Soriano &San Segundo, 2009), gfp expression did not affect patho-genicity nor the ability of the M. oryzae fungus to sporu-late on the infected leaf. The infection process wasfollowed visually and microscopically up to 6 dpi.The resistant cv. TeQing did not develop disease symp-

toms on its leaves, not even when the highest inoculumdose was used (106 spores mL�1; Fig. 1a). Saber andKaty exhibited small necrotic spots, if any, appearing astiny, pinpoint dots at the inoculated regions that did notfurther expand on the leaf surface (106 spores mL�1).However, cvs Saber and Katy never developed typicalblast lesions on their leaves. Representative results areshown in Fig. 1b,c. Under the same experimental condi-tions, leaves from Kanto 51 exhibited lesions which weremost common at the distal portions of the inoculatedleaves (Fig. 1d). By contrast, Kanto 51 exhibited resis-tance to blast in the field (Tacconi et al., 2010). Previousstudies have shown that the detached leaf assay enhancessusceptibility to blast infection compared with inocula-tion of whole plants (Berruyer et al., 2006). This factmight explain the different responses observed in Kanto51 depending on the method used for inoculation,namely the detached leaf assay (present work) versusinfection at the whole-plant level (Tacconi et al., 2010).When infected with the gfp-M. oryzae isolate, the sus-ceptible genotypes Senia, Vialone Nano and Maratellishowed severe symptoms of infection. With time, theselesions developed into typical blast lesions (Fig. 1e–g).Leaf lesions were also observed by fluorescent micros-

copy, which allowed the in planta detection of the fungalmycelium. As depicted in Fig. 1e–g (lower panels), theinoculated regions showed bright GFP fluorescence. Alarge number of fluorescent hyphae grew and sporulatedon the leaf surface in cvs Vialone Nano and Maratelli(Fig. 1f,g; lower panels).The observed susceptibility to M. oryzae infection of

the various rice genotypes was further assessed by imageanalysis. The percentage of leaf area affected by blastlesions at 3 and 6 dpi was determined. In agreementwith the visual inspection of infected leaves, no evidentblast lesions developed in infected leaves of cvs Saber

and Katy (Fig. 2a,b). The small percentage quantified byimage analysis at 6 dpi in Katy leaves (and to a lesserextent in Saber leaves) account for the small necroticspots, but not for blast lesions. Presumably, the appear-ance of these necrotic spots in Saber and Katy wouldprevent tissue colonization which, in turn, would con-tribute to the resistance phenotype that is observed inthese cultivars. Although infected leaves of Kanto 51showed visible lesions at 3 dpi at the highest inoculumdose (106 spores mL�1), these lesions did not developwith time (3 and 6 dpi; Fig. 2c). In contrast to Kanto51, a direct correlation between lesion size and inoculumconcentration was observed in infected leaves of the sus-ceptible cvs Senia, Vialone Nano and Maratelli, the areasof these lesions increasing with time (Fig. 2d–f).

CLSM analysis of susceptible rice cultivars

Confocal microscopy was used to investigate in detailthe early stages of the infection process in the susceptiblerice genotype Vialone Nano. Magnaporthe oryzae sporeswere easily visualized on the leaf surface and started togerminate by 3–6 h after inoculation (Fig. 3a). Typically,each spore produced a polarized germ tube, whichemerged from the apical cell of the conidium (Fig. 3b).Penetration structures were identified by localized hyphalswellings corresponding to appressoria (Fig. 3b,c). Afterpenetration into the host tissue, thin filamentous primaryhyphae grew in the cell lumen and subsequently differen-tiated into bulbous and branched secondary hyphae.Confocal images illustrating the dimorphism of primaryand internal hyphae are shown in Fig. 3c–f. A closeinspection of CLSM images of the infected leaves alsorevealed specific locations at which hyphae constricted(Fig. 3e,f; arrowheads). These specialized hyphae, alsoreferred to as invasive hyphae pegs, allow the cell-to-cellmovement of the fungus. Bulbous infection hyphae werealso detected inside the guard cells (Fig. 3g). By 3 dpi,contiguous mesophyll cells were invaded by hyphaeforming green fluorescent lines along the leaf (Fig. 3h,i).By this time, patches of mycelial network had alsoformed on the surface of the leaf (Fig. 3j).

Confocal analysis of resistant rice cultivars

Leaves of cvs Saber and TeQing (Pib gene) were inocu-lated with spores of the gfp-expressing M. oryzae isolateand examined by CLSM during the early stages of fungalgrowth. Results are presented in Figure 4. Magnaportheoryzae spores germinated freely, but the majority of thegermlings produced highly abnormal germ tubes, withconstrictions and bulges distributed along them. Thespore and germ tube cells of germlings growing on Saberand TeQing were highly vacuolated (Fig. 4a–c) comparedto germlings growing on the susceptible cv. VialoneNano. Frequently, more than one germ tube formed fromthe same spore (Fig. 4d–f; arrowheads). Althoughappressoria formed on the leaf surface, there was novisible evidence of penetration events, either in Saber or

Plant Pathology (2013) 62, 1030–1037

1032 L. Campos-Soriano et al.

TeQing. Infectious hyphae were not detected inside hostcells, not even 60 h after inoculation. These observationssupport that growth and development of germlings isseverely impaired in leaves of Saber and TeQing whichmight, in turn, prevent penetration into the host tissue.

Discussion

Blast disease, caused by M. oryzae, is one of the mostserious diseases of rice worldwide. This is apparently the

first study describing blast infection in susceptible andresistant rice cultivars using a gfp-expressing isolate ofM. oryzae. The infection process was followed at themacroscopic level up to 6 dpi (necrotrophic stage). Thegfp-expressing isolate was also used to examine, bymeans of CLSM, the occurrence of reactions in the path-ogen upon inoculation of resistant rice cultivars. In allcases, detached leaves were locally inoculated withincreasing concentrations of M. oryzae spores. Thedetached leaf assay has proven to be a convenient

(a)

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TeQing

Saber

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Seniai ii

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Figure 1 Infection of detached rice leaves with spores of the gfp-expressing Magnaporthe oryzae isolate. Leaf symptoms were observed by light

and fluorescent microscopy (upper and lower panels, respectively, in d–g). Pictures were taken at 6 days post-inoculation with M. oryzae spores

(106 spores mL�1). (a–c) Cultivars TeQing (a), Saber (b) and Katy (c) showed no lesions, but small necrotic spots were visible at inoculated areas

in Saber and Katy; (d) Kanto 51 showed some lesions, mainly at the distal regions of inoculated leaves; (e) Senia showed several lesions with

necrotic areas and very little initial sporulation. (f–g) Vialone Nano (f) and Maratelli (g) showed extensive necrotic lesions in which the fungus formed

a bright fluorescent network with active sporulation. Representative results from one of four experiments that produced similar results are shown.

Plant Pathology (2013) 62, 1030–1037

M. oryzae development in resistant and susceptible rice 1033

method for the analysis of the early in planta growthstages of the rice blast fungus, yielding highly uniformand reproducible infection in the rice leaf (Berruyeret al., 2006). Nevertheless, detached leaves showenhanced susceptibility to the rice blast fungus comparedwith inoculation of whole plants (Berruyer et al., 2006).This would explain the difference between the resultsobtained in this study, where nectrotic lesions wereobserved on detached leaves of Kanto 51 (Pik gene), andthose previously obtained with artificial inoculation ofwhole Kanto 51 seedlings, which showed resistance (Tac-coni et al., 2010). It is also true that blast lesions in Kan-to 51 leaves only developed at the highest inoculum dose(106 spores mL�1) in the present work, and that thisinoculum dose was much higher than that used to spray-inoculate whole seedlings (104 spores mL�1; Tacconiet al., 2010). Although M. oryzae-infected Kanto 51leaves developed necrotic lesions, sporulation in theselesions was relatively low. Contrary to this, a densehyphal mycelium covered blast lesions in Vialone Nanoat 3 dpi, and by 6 dpi the fungus was sporulating abun-dantly on these lesions.Confocal laser scanning microscopy also revealed fun-

gal penetration into the epidermal host cells and coloni-zation of mesophyll cells in Vialone Nano. The primary

hyphae filled the first-invaded cells, and moved into theneighbouring cells, presumably via plasmodesmata, afterproducing highly constricted hyphae that crossed the cellwall (Kankanala et al., 2007). The primary hyphae dif-ferentiated into bulbous hyphae that were thicker thanthe primary ones, thus confirming that the fungus hadentered the necrotrophic phase. This morphology andbehaviour has been already described in other compatiblerice–M. oryzae interactions (Kankanala et al., 2007;Campos-Soriano & San Segundo, 2009).Whereas the process of infection by M. oryzae in sus-

ceptible rice cultivars is documented in the literature, fewstudies have addressed the impact of R gene action onthe early stages of fungal growth in resistant rice culti-vars. Most blast disease resistance genes encode nucleo-tide-binding site–leucine-rich repeat (NBS–LRR) proteinswith a predicted intracellular location. The predictedintracellular location of the cognate resistance gene prod-ucts implies that the fungal virulence factors that interactwith these resistance gene products enter into the cyto-plasm of plant cells. For instance, the Pita gene wasshown to mediate gene-for-gene resistance againstM. oryzae isolates that express avirulent alleles of AVR-Pita (Bryan et al., 2000). It has been proposed thatAVR-Pita is delivered into the cytoplasm of the rice cell

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Figure 2 Percentage of leaf area affected by blast lesions in leaves of resistant and susceptible rice cultivars, as determined by the detached leaf

assay 3 and 6 days post-inoculation (dpi) using image analysis software ASSESS v. 2.0 for plant disease quantification (Lamari, 2008). Four

independent experiments were carried out. For each experiment, three leaves were inoculated with each of three inoculum doses: 104 (black bars),

105 (grey bars) and 106 spores mL�1 (white bars), with five inoculations per leaf. Means and standard errors are indicated.

Plant Pathology (2013) 62, 1030–1037

1034 L. Campos-Soriano et al.

where it binds to the Pita protein to initiate a Pita-medi-ated defence response (Jia et al., 2000). However, despitethe large number of resistance genes that have been iden-tified and mapped in the rice genome, only a few blastresistance genes have been functionally characterized(e.g. Pib, Pita, Pik and Pi54) and the prospect for dura-ble control of rice blast based on the development ofresistant genotypes carrying R genes still represents asubstantial challenge.The rice cultivars examined in this study were TeQing

and Saber (Pib gene), Katy (Pita2 gene) and Kanto 51(Pik gene). The Pib gene (distal end of the long arm ofchromosome 2), was shown to confer a high level ofresistance to most Japanese, some Chinese and all Euro-pean isolates tested (Roumen et al., 1997; Wang et al.,1999). Rice breeding programmes incorporated Pib fromthe Chinese cultivar TeQing into Saber (McClung et al.,2004). As for the Pita gene (chromosome 12), it confersresistance to most of the European and two AfricanM. oryzae races (Roumen et al., 1997; Jia, 2009). ThePita resistance gene from the Vietnamese landrace Tetepwas incorporated into cv. Katy (Moldenhauer et al.,1990). This R gene is also considered an important

source of resistance to blast, and has been effectivelydeployed to prevent blast in the southern USA for over adecade (Moldenhauer et al., 1990; Jia et al., 2004).Finally, the Pik locus (long arm of chromosome 11)includes at least five blast R genes (Pik, Pik-m, Pik-p,Pik-h and Pik-s) (Ashikawa et al., 2008; Wang et al.,2009). With the exception of Kanto 51, the resistant cul-tivars in the present study (TeQing, Saber and Katy) didnot develop blast lesions.To gain further insights into the basis of blast resis-

tance, a detailed study of the initial stages of the interac-tion of M. oryzae with the resistant cvs Saber andTeQing was carried out by CLSM, focusing on sporegermination and germling development. This revealedthat germling growth was markedly and adverselyaffected in the resistant cultivars. Even though sporesgerminated on the leaf surface of Saber and TeQing, theyproduced morphologically abnormal germlings. Penetra-tion could not be detected in any of these resistant geno-types. Presumably, the abnormal appearance of germtubes, then becoming vacuolated, and the apparent lackof penetration would reflect R-gene activity. If so, theseobservations might be indicative that, at least in part, the

6 hpi 6 hpi 30 hpi(a) (b) (c)

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Figure 3 Confocal laser scanning microscopy of leaves from susceptible rice cv. Vialone Nano inoculated with spores of the gfp-expressing

Magnaporthe oryzae isolate. (a, b) Germination of conidia and appressorium formation on the leaf surface 6 h post-inoculation (hpi). (c–f) Hyphae

developing into invaded tissue, showing the dimorphic growth pattern typically observed during the biotrophic (thin hyphae) and necrotrophic (thick

and bulbous hyphae) stages of leaf colonization by M. oryzae; (e, f) hyphal constrictions at sites of invasion of contiguous mesophyll cells

(arrowhead). (g) Bulbous secondary hyphae invading a guard cell 3 dpi. (h, i) Mesophyll cells being filled with highly invasive hyphae. (j) Hyphal

network growing on surface of leaf tissue (3 dpi). Projection images are shown. Key: sp, spore; gc, guard cell; gt, germ tube; ap, appressorium; ep,

epidermal cells, me, mesophyll cells. Scale bars = 20 lm (a, i), 10 lm (b, c, d, e, j) and 5 lm (f–h).

Plant Pathology (2013) 62, 1030–1037

M. oryzae development in resistant and susceptible rice 1035

resistance observed in Saber and TeQing results frominterference in germling growth and/or the failure of thegerm tube to differentiate into a functional appressorium.The increased vacuolation that accompanies morphologi-cal defects in germ tube cells might reflect a misregula-tion in processes mediating turgor generation in theappressorium, which then cannot be translated into phys-ical force for penetration into the host tissue. Addition-ally, the possibility that recognition of the pathogen bythe host cell leads to the production of chemicals or fac-tor(s) being released to the leaf surface and disruptinggermling development should be considered. Alterna-tively, host cell wall modification and creation of struc-tural barriers in the rice cells may well cause the host toresist attack by the fungus. Whatever the explanation is,it is intriguing to observe that these cytological reactionsof M. oryzae cells in resistant cultivars occur in theabsence of host cell penetration. Disruption of germlingdevelopment on the leaf surface might be part of theresistance mechanisms induced by Pib functioning. In

other studies carried out on typical gene-for-gene interac-tions, major blast resistance genes have been shown toprevent disease by blocking fungal growth after the path-ogen penetrates into the host tissue and begins to growwithin rice cells (i.e. the interaction of the Pita and avi-rulence gene products in the cytoplasm of the plant cell;Jia et al., 2000).On the other hand, elegant studies by Veneault-Four-

rey et al. (2006) provided evidence that the formation ofa functional appressorium requires, sequentially, thecompletion of mitosis, nuclear migration, and death ofthe fungal spore. Autophagic cell death of the fungalspore was found to be a prerequisite for infection. Thus,it is possible that impairment or failure in orchestratingthese processes might contribute to resistance in cvsSaber and TeQing. Clearly, further studies are needed toshed light on the mechanisms responsible for theobserved abnormalities in M. oryzae germlings growingon the leaf surface of these resistant rice cultivars.Collectively, the results presented here illustrate the

usefulness of the combination of fluorescently labelledfungal isolates and CLSM for studies on rice–M. oryzaeinteractions. The use of gfp-expressing M. oryzae will bevaluable for identifying resistant genotypes in rice germ-plasm in future breeding programmes and for evaluatingthe effectiveness of R-gene-mediated resistance. Under-standing processes and resistance mechanisms operatingin the rice plant during its interaction with the rice blastfungus is of paramount importance for the developmentof effective and durable strategies to manage rice blastdisease.

Acknowledgements

LCS was a recipient of a predoctoral fellowship from theGeneralitat de Catalunya. We are grateful to A. God�ofor her collaboration in parts of this work. This workwas funded by grant BIO2009-08719 from MINECOand the Proyecto Intramural 200420E613 from CSIC toBSS, the Consolider-Ingenio CSD2007-00036 to CRAG,the VALORYZA project (DM 301/7303/06 Ministerodelle Politiche Agricole, Rome, Italy) to EL, and the EUco-funded project EURIGEN (049 AGRI GEN RES). Wealso thank the Xarxa de Referencia en Biotechnologiaand SGR (Support to Research Groups from the Ag�enciade Gesti�o d’Ajuts Universitaris i de Recerca) from theGeneralitat de Catalunya for substantial support. ELacknowledges a CRA grant as visiting scientist at CRAG,Barcelona.

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6 hpi 6 hpi

sp t

ap

gtapsp

g

6 hpi 6 hpi

6 hpi 6 hpi

gt sp

sp apgt

ap

gt

ap

sp

sp

gt

(a) (b)

(c) (d)

(e) (f)

Figure 4 Confocal laser scanning microscopy of leaves from the

resistant rice genotypes Saber (a,d) and TeQing (b, c, e, f) inoculated

with spores of the gfp-expressing Magnaporthe oryzae isolate, 6 h

post-inoculation (hpi). Germ tubes showed abnormal growth and

vacuolization of spore and germ tube cells was frequently observed,

as well as spores forming a second germ tube (arrowheads in d–f).

Projection images are shown. Key: sp, spore; gt, germ tube; ap,

appressorium. Scale bars = 10 lm (a–d), 20 lm (e, f).

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Supporting Information

Additional Supporting Information may be found in the online version of

this article.

Figure S1. Confocal laser scanning microscopy of leaves from the sus-

ceptible rice cultivar Vialone Nano inoculated with spores of the gfp-

expressing Magnaporthe oryzae isolate. Same images as in Fig. 3 using

colours suitable for colour-blind readers (green-blue colours).

Figure S2. Confocal laser scanning microscopy of leaves from the resis-

tant rice genotypes Saber and TeQing inoculated with spores of the gfp-

expressing Magnaporthe oryzae isolate. Same images as in Fig. 4 using

colours suitable for colour-blind readers (green-blue colours).

Plant Pathology (2013) 62, 1030–1037

M. oryzae development in resistant and susceptible rice 1037