Searching for Moniliophthora perniciosa pathogenicity genes
Introduction
Witches’ broom is a severe disease of Theobroma cacao (cacao) caused by the basidiomycete Moniliophthora perniciosa (Stahel) (Aime & Phillips-Mora 2005), formerly Crinipellis perniciosa. This fungus occurs naturally in the Amazon basin infecting cacao and related species, and some weedy solanaceous and woody liana vine species (Griffith et al. 1994). The sequential introduction of witches’ broom disease in various producing regions in South America and Caribbean has devastated local cacao industries (Andebrhan et al. 1999). The ultimate method of witches’ broom control will be through host genetic resistance (Purdy & Schmidt 1996). However, the most widely used source of resistance (‘Scavina 6’) has suffered increasing infection symptoms in Bahia, Brazil, possibly associated with fungal genetic variability (Paim et al., 2006, Albuquerque et al., 2010).
Moniliophthora perniciosa is a hemibiotrophic pathogen, with contrasting mycelial morphology and behavior during the biotrophic and necrotrophic phases (Griffith et al. 1994). The biotrophic phase starts with basidiospore germination, followed by stomatal penetration and apoplast colonization, without any specialized structure (Frias et al. 1991). The pathogen generally infects cacao meristematic tissues (shoots; flower cushions; single flowers; and developing pods), inducing a range of symptoms depending on organ infected and stage of development (Purdy & Schmidt 1996). Hypertrophic growth of infected shoots (‘brooms’) is the most dramatic symptom, arising typically 15 d after infection (Purdy & Schmidt 1996). External symptoms generally evolve to tissue necrosis, from where basidiocarps emerge (Purdy & Schmidt 1996). Usually, the biotrophic phase has been referred to as associated with monokaryotic mycelium, but there are contradictory descriptions about the nuclear conditions of the hyphae during that stage (Purdy & Schmidt 1996), and both mycelia phases may occur at the same time in colonized living tissues (Ceita et al. 2007). Causal stimuli of mycelia phase change in planta are presently unknown, and uninucleate and binucleate mycelia have been obtained in vitro (Meinhardt et al. 2006).
The mechanisms of pathogenesis of M. perniciosa are largely undefined. Search for genes necessary for disease development (‘pathogenicity genes’) by exploring databases for sequences coding for lytic, detoxifying or for toxin biosynthesis enzymes is limited by the restricted similarity between pathogenicity genes from distinct organisms (Idnurm & Howlett 2001). Large scale sequencing of host–pathogen interaction expression sequence tag (EST) libraries could identify novel pathogenicity genes, but the low representation of pathogen transcripts in such libraries would require an exhaustive number of clones to be sequenced (Guilleroux & Osbourn 2004), especially for pathogens with reduced colonization, such as M. perniciosa (Leal et al. 2007). Searching for putative pathogenicity genes in EST libraries obtained from in vitro grown M. perniciosa mycelia at monokaryotic (‘biotrophic-like’) and dikaryotic phases, together with microarray analyses, disclosed potential candidates (Rincones et al. 2008). Among the identified sequences, putative pathogenicity genes, such as necrosis and ethylene-inducing proteins and a ceratoplatanin, together with genes associated with nitrogen- or carbon-catabolite repression were described, but not functionally evaluated in the host–pathogen interaction (Rincones et al. 2008).
An alternative approach to identify potential pathogenicity genes consists in searching for sequences induced in mycelia grown in vitro under stressful conditions, such as nitrogen (N) and/or carbon (C) limitation; photostimulation; or modification of media pH (Ehrenshaft and Upchurch, 1991, Prusky and Yakoby, 2003, Bolton and Thomma, 2008). Some pathogenicity genes have been shown to be up-regulated when mycelia were grown in vitro under N limitation (Bolton & Thomma 2008). Further, it has been hypothesized that the host environment initially colonized by pathogens might be deprived of N (Bolton & Thomma 2008), or some essential amino acids (Solomon et al. 2003) functioning as a stimulus for induction of pathogenicity genes in planta. Based on this hypothesis, pathogenicity candidate genes have been identified from various pathogens, including Gibberella zeae (Trail et al. 2003) and Magnaporthe grisea (Donofrio et al. 2006). Production of fungal virulence factors, such as toxins or low molecular weight proteins, might occur in vitro under N limitation (Stephenson et al. 2000) or light (Ehrenshaft & Upchurch 1991), and part of the genes induced in vitro might be induced in planta (Bolton & Thomma 2008).
The objectives of this work were to recognize genes differentially expressed in M. perniciosa cultivated in vitro mediated by N limitation and light to identify putative pathogenicity genes, and to investigate, after proper validation, their expression at the initial asymptomatic stage of infection in cacao plants. The M. perniciosa sequences were derived from a cDNA library obtained by suppression subtractive hybridization, enriched for genes expressed under N limitation and photostimulation. From eight transcripts selected to be validated representing categories containing either potential genes or functions associated with pathogenicity, seven were confirmed with differential expression between mycelia grown in vitro on light under contrasting N conditions. Among the seven validated genes, the expression of six was detected in planta at distinct moments during infection: one group was more expressed at the initial phase of colonization, whereas the other was more prominent at advanced stage of the asymptomatic phase of infection. These six genes presented a more intense expression than a fungal housekeeping gene at the initial stage, when the first host defence response typically occurs, suggesting that these induced genes might be involved with pathogen protection and adaptation during host response.
Section snippets
Moniliophthora perniciosa inoculum and plant material
Dry infected cacao shoots (‘dried brooms’; 50 cm) were collected at the ‘Estação de Recursos Genéticos do Cacaueiro José Haroldo’, Marituba, PA, Brazil (1°12′S; 49°13′W). Basidiocarps were induced by exposing brooms to alternating wet–dry cycles (12 h). Storage spore suspension was prepared in 16 % glycerol in 0.01 M 2-morpholinoethanesulfonic acid (MES) (Frias et al. 1995). The isolate ERJOH-1 was maintained on Potato-Dextrose-Agar (PDA) medium at room temperature. Clonal plants of the witches’
Establishing in vitro growth conditions for M. perniciosa
Fungal cultures were grown under distinct N concentrations (4 or 100 mM N), supplied either as ammonium or nitrate, and exposed to light (25 μmol m−2 s−1) for 12 h or kept in darkness, and evaluated after 30 d to define the most contrasting growth conditions. Mycelia exhibited the maximum growth under the highest N dose, independent of the source, and especially in the dark (Table 1). At the low ammonium concentration under light, the mycelia grew poorly, but sufficiently to allow RNA extraction,
Discussion
Mimicking stressful conditions in vitro has been a recognized approach to search for putative pathogenicity genes active during early host tissue colonization (Donofrio et al. 2006). Pathogenicity genes may be induced by limited nitrogen availability, whereas phytotoxin production may be stimulated by nutrient limitation and/or light (Ehrenshaft and Upchurch, 1991, van den Ackerveken et al., 1994, Bolton and Thomma, 2008). To identify potential pathogenicity genes in the cacao pathogen
Acknowledgments
This manuscript is dedicated to the memory of Silmara Moriya. The authors wish to thank FAPESP for financial support (03/11483-0; 07/07175-0) and CEPLAC for technical support. GAL, PSBA, and AF were recipients of CNPq fellowships. Technical assistance by Edivaldo Pimentel is greatly appreciated.
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