Cellular and molecular characterization of resistance to B. sorokiniana in bread wheat
Sundeep Kumar1, Patrick Schäfer2, Carin Janso2, Gregor Langen2, Karl-Heinz Kogel2, Bhakti Rana1, Manoj K. Yadav1 and A. K. Joshi3
1Department of Biotechnology, Sardar Vallabh Bhai Patel University of Agriculture & Technology, Modipuram, Meerut, 250110
2Institute of Phytopathology and Applied Zoology, Giessen, Germany
3Department of Genetics and Plant Breeding, BHU, Varanasi
Corresponding author: Sundeep Kumar
E-mail: sundeepksharma@rediffmail.com
Abstract
Spot blotch disease of wheat has now been considered as one of the major constraints in wheat growing regions specially in South East Asia and Latin American countries where warm humid conditions persist during wheat crop season (Kumar et al. 2002). Due to tremendous breeding efforts, new partially resistant lines have recently been released. However, lack of complete resistant genotypes and insufficiency of knowledge about the resistance mechanism(s) still hamper sustained breeding efforts. We comparatively investigated the interaction of B. sorokiniana with moderate resistant genotype Yangmai 6 and susceptible genotype Sonalika. Cytological data have been recorded at different time points about the changes occurring at cellular level viz., cell wall-associated defense (CWA), hypersensitive reaction (HR), and post-penetration hypersensitive reaction (PPHR). The molecular studies aim at identification and functional analysis of genes, whose expression is getting influenced by the onset of B. sorokiniana infection. On the basis of macroarray spotted with 1,536 cDNA fragments, hybridized with probes prepared from barley leaves at two time points after fungal infection, 52 candidate genes were identified either up- or down regulated. Five randomly selected candidate genes of this collection were further verified by RT-PCR and altered candidate gene expression was compared at mRNA transcriptional level in response to B. sorokiniana infection in both genotypes. The candidate genes were actin, ABC transporter related gene, ferritin, Mg-chelatase subunit XANTHA-F and RNA binding protein Rp 120. The putative role of the candidate genes will be discussed.
Key words: Wheat, Bipolaris sorokiniana, macroarray, RT-PCR, candidate genes
Introduction
Wheat is considered as most widely grown and consumed food crop of the world and is expected to be the staple food of around 35% of the world population (Pingali ed. 1999). The present wheat production is about 560 million tonnes and to feed world ever-growing population in 2020 we have to produce from 860 to 1040 million tonnes with 1.6 to 2.6% annual growth rate. To achieve the target, it is essential to keep the wheat crop free from various biotic stresses including spot blotch (B. sorokiniana), which are limiting wheat production in different parts of the world (Mathur and Coufer 1993). Spot blotch has now been emerged as one of the major production constraint in the wheat growing areas particularly in South East Asia and Latin American countries where nearly 12 million hectares of land under cultivation are affected (Nagarajan and Kumar 1998). The pathogen affects almost all the crops belonging to Graminace family, though the chances of migration of isolate of one crop to other crop are remote (Bakonyi et al. 1997; Pandey et al. 2005). B. sorokiniana causes foliar spot blotch, root rot, black point on grains, head blight and seedling blight of wheat and barley (Kumar et al. 2002). Estimates of yield losses due to spot blotch are reported to vary from 15.5 to 19.6% (Dubin and Van Ginkel 1991), 20 to 80% (Duveiller and Gilchrist 1994), and may be upto 100% under severe infection conditions (Mehta 1994). An integrated approach including right cropping system, use of fungicides and introduction of new resistant varieties can provide effective control of spot blotch under field conditions (Joshi and Chand 2002). Bipolaris sorokiniana is a hemibiotrophic fungus having both biotrophic and necrotrophic phases. The biotrophic growth phase is confined to a single epidermal host cell that is invaded by a network of infected hyphae, while the necrotrophic growth phase is characterised by invasion of the mesophyll tissue and host cell death (Kumar et al. 2002; Schäfer et al. 2004). Plant resistance against any pathogen can be classified broadly into two classes non-host resistance and host resistance. The non-host resistance is found in all cultivars of a plant species to all races of a certain pathogen. Non-host resistance obviously relies on a complex genetic control and implies a genotype of divergent defence components whose existence or induction does not depend on known resistance genes (Heath 2000). The molecular basis of non-host resistance includes synthesis of secondary metabolites with antimicrobial activity, hypersensitive reaction (HR), production of phytoalexins, extra cellular accumulation of reactive oxygen intermediates (ROI) and the activation of defence related genes especially, cell wall-associated defence (CWA) mechanisms, and post-penetration hypersensitive reaction (PPHR). Host resistance specific to genotype of the host, starts to work when pathogen over come non-host resistance devices. The increasing threat of spot blotch calls for serious efforts to understand various dimensions including cytological and molecular basis of the resistance mechanism to breed resistant genotypes. However, to reveal the resistance mechanism at cytological basis, still a detailed cytological study is needed to know about different components, which are directly or indirectly related to resistance mechanism. The aim of molecular studies about candidate genes along with cytological studies was just to bring valuable information to have the better understanding of resistance mechanism at cytological as well as molecular level.
Materials and Methods
Genotypes and pathogen
Moderately resistant genotype Yangmai 6 and susceptible genotype Sonalika were used in the study. Seeds of the given varieties were obtained from International Maize and Wheat Improvement Centre (CIMMYT), Mexico and the Directorate of Wheat Research (DWR), Karnal, India, respectively. Informations about isolation and origin of Bs culture used in the study are given in Kumar et al. (2001). For the spray, bits from monoconidial cultures were placed on the filter paper in SNA media and incubated at room temperature under a 12 h-photoperiod. After 7 days, petri plates were flooded with the solution of distilled water with Tween 20 in the ratio (5000:1). Conidia were scraped out from the surface with a spatula and filtered through two layered muslin cloth before inoculation.
Microscopic analysis
For cytology, About 6 cm long leaf segments of flag leaves from 15-day-old plants were placed on whatman filter paper and incubated with spore suspension containing 50,000 spores/ml. After spraying with spore suspension, leaf segments were placed on 0.5% water agar (40 mg/l bengimidazol) in a plastic dish (>95% RH) closed tightly with a lid. Plates were incubated in a growing chamber (24˚C day/night and 16-h photoperiod). The leaf segments were evaluated according to the stipulated time point kinetics viz., 20, 40 and 60 hours respectively. Calcofluor has been used for cell and hyphal wall detection (Rohringer 1977; Schäfer et al. 2004) and facilitates the location of fungal penetration sites in leaf tissue. For better contrast of defense reactions, histochemical detection of H2O2 was carried out by an endogenous peroxidase-dependent in situ staining procedure with 3,3-diaminobenzidine (DAB, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) (Thordal-Christensen et al. 1997). Leaves were infiltrated with 1 mg/ml DAB dissolved in water (pH3.8, HCl) and injected into wheat first leaves 2 h before fixation. Leaf fixation and microscopy were conducted as described by Hückelhoven and Kogel (1998). Whole cell and sub cellular autoflorescece was observed under epifluorescence microscopy (Kumar et al. 2002; Schäfer et al. 2004). Moreover, calcofluor stained hyphae were observed by florescence microscopy. Four leaf segments and 60-70 interaction sites on each leaf segment were elucidated in two independent experiments.
Identification and functional analysis of candidate genes
For molecular analysis, plants were grown in a growth chamber at 24˚C/18˚C (day/night cycle), 60% RH and a photoperiod of 16h (240 µmol m-2S-1 photon flux density). 15 days old plants were inoculated by spraying a spore suspension containing 100,000 spores/ml. An epidermis-specific cDNA library derived from barley leaves treated with culture filtrate of B. sorokiniana was synthesized. On the basis of macroarray spotted with 1,536 cDNA fragments, hybridized with probes prepared from barley leaves at two time points after fungal infection, 52 candidate genes were identified either up or down regulated. Five randomly selected candidate genes of this collection were further verified by RT-PCR and altered candidate gene expression was compared at mRNA transcriptional level in response to B. sorokiniana infection in wheat.
RNA extraction and cDNA synthesis
Total RNA was extracted from 8 cm long leaf segment of flag leaf using RNA extraction buffer (Peq lab, Erlangen, Germany). RNA was extracted from the Bs treated leaf samples collected at the different time points viz., 0, 8, 24, 48 and 72 h after inoculation in parallel with corresponding mock treated leaf segments (1:5000; Tween 20:water). RNA quantity and quality was measured at 260 and 280 nm by UV-spectrophotometer (DU7400, Beckman) and in dehydrated formaldehyde gel electrophoresis, respectively. RT-PCR kit (MBI-Fermentas, Germany) was used for the synthesis of cDNA following the manufacture’s instructions as written in the literature provided with Reverse Transcriptase enzyme. 4 µg RNA was used to construct the first strand cDNA by using Reverse transcriptase and oligo-DT primers. In the RT-PCR analysis, barley ESTs corresponding to these genes were used in the construction of primers for RT-PCR analysis (Table 1).
Results
Initial field screening
Both the varieties had been intensively screened under the field condition by the first author at the Agricultural Research Farm, Banaras Hindu University, Varanasi (India) during 1999 to 2001. Yangmai 6 was showing less disease severity even at early dough stage (growth stage 83; Zadoks et al. 1974) (data not shown). In addition to this, both varieties were also screened under controlled conditions by producing artificial epiphytotic condition at Institute of Phytopathology and Applied Zoology, JLU, Giessen. Detached leaf segments of the youngest and 2nd youngest leaf from 15-day-old seedlings were screened under artificial epiphytotic condition to access the level of disease severity. Sonalika was showing more number of spot blotch lesion per leaf segment in comparison to moderate resistant genotype Yangmai 6.
Microscopic analysis
Fungal penetration through anticlinal cell walls and invasion of the mesophyll tissue starting from the epidermal layer were similar to what was found in barley-B. sorokiniana interaction in earlier studies (Kumar et al. 2001; Schäfer et al. 2004).
Fungal penetration into epidermal cells was termed “successful penetration” and invasion of mesophyll tissue “successful infection”. Higher level of infection was observed in susceptible cultivar Sonalika. All defense responses were grouped into two broad classes i.e., Pre defense and post defense responses. Cell wall appositions (CWA) and hypersensitive reaction (HR) were considered under pre defense responses, while post penetration defense reaction (PPHR) was taken as post defense response. Higher frequency of non-penetrated epidermal CWA was observed in Yangmai 6 at all time points (20, 40 and 60 hai), while Sonalika showed higher frequency of penetration (Fig. 1).
The frequencies of non-penetrated epidermal cells that underwent an HR were analyzed. However, a rapid decrease at 40 and 60 h after inoculation was observed in moderate resistant genotype Yangmai 6. In the early hours (20 hai), the number of Post Penetration Hypersensitive Reaction (PPHR) sites was not too different in both varieties but at 40 hai, Yangmai 6 was showing comparatively higher level of PPHR in comparison to Sonalika (Fig. 1). The fungal spreading was observed to be correlated with H2O2 accumulation. The amount of H2O2 in the leaves showed a correlation with necrosis. Higher H2O2 accumulation was recorded in susceptible genotype Sonalika than Yangmai 6. Autofluorescence observations were closely related with subcellular and whole cell accumulation of H2O2 as visualized after the injection of 3,3’ diaminobenzidine (DAB) into the excised leaf segments (Kumar et al. 2001).
Macroarray analysis with Bipolaris sorokiniana
To study the mechanism of resistance against spot blotch invasion, molecular study was began with macro array analysis based on barley crop. 52 genes were identified either up or down regulated at 20 and 40 h after inoculation with Bs culture. Reliable data were obtained by repeating array hybridization three times. Each clone was spotted two times on each array and mean values of the corresponding spots were used in signal transduction. The putative function of each gene was defined according to Blast X2 results (Schäfer 2004). Five of 52 randomly selected candidate genes were further verified by RT-PCR and altered candidate gene expression were compared at mRNA transcriptional level in response to B. sorokiniana infection in moderate resistant genotype Yangmai 6 and susceptible genotype Sonalika of wheat (Table 2).
RT-PCR analysis of candidate genes
In comparison to control, gene from ABC1 family was showing down regulation at 8, 24 and 48 hai in Bs treated samples in both genotypes, Sonalika and Yangmai 6. However, at 72 hai bands of control and treated one were showing equal intensity in agarose gel having ethidium bromide (Fig. 2). The RT-PCR results were in accordance to Macro-array analysis based on barley.
Mg chelatase subunit XANTHA-F gene, observed to be down regulated in Macro-Array analysis was showing clear down regulation at 8 hai in both cultivars of wheat. However, an up regulation after 3 days (72 hai) was observed in Sonalika.
In case of actin, it was down regulated at all defined time points viz., 8, 24, 48 and 72 h after inoculation. However, it is seen that down regulation was more readable in susceptible cultivar Sonalika. Actin gene was considered to be down regulated in Macro Array analysis with the infection of B. sorokiniana.
Ferritin gene was observed up regulated in both varieties at 72 h after inoculation in comparison to control. Moreover, it was more up regulated in susceptible cultivar Sonalika than moderate resistant genotype Yangmai 6 with Bs culture filtrate. In both varieties, a down regulation at 24 h after inoculation was also noticed.
In contrast to Macro array analysis based on barley, RNA binding protein Rp 120 was showing down regulation at 48 and 72 h after inoculation in wheat cultivar Sonalika compared to control (mock treated plants). However, slight up regulation was noticed at 48 and 72 h after inoculation in Yangmai 6.
Discussion
Cytological analysis
The purpose of characterization of disease resistance at cytological level was to build up a plate farm for molecular studies. Wheat resistance to B. sorokiniana is partially attributed to penetration resistance due to papilla-like CWA or early HR of non-penetrated cells (Fig. 1). Since, B. sorokiniana is a hemibiotroph (Kumar et al. 2002; Schafer et al. 2004), it takes food first from attacked living cell before the deposition of CWA and then from dead cell. Likewise, HR-associated cell death might be an inadequate measure to stop necrotrophic fungus (Kumar et al. 2001; Kumar et al. 2002). One of the acceptable reasons in the susceptible genotype might be that the activation of such defence responses often occurs so late that host fail to restrict ingress of the pathogen. Effective penetration resistance is closely associated with the local generation of H2O2 (Kumar et al. 2001; Shäfer et al. 2004). This H2O2 leads the necrosis of cells in the surroundings of pathogen and thus, localize the pathogen attack. Since, fungal penetration attempts into the epidermal cells were prevented as such at the site of H2O2 accumulation, therefore role of H2O2 in the inaccessibility of epidermal cells cannot be overruled. Moreover, post-penetration HR encapsulated the infected hyphae from first penetrated cell and therefore, localised the pathogen. Cell death at the early (epidermal) growth stage results in fungal arrest, while cell death at a later stage (mesophyll) might be either a prerequisite or a symptom of successful fungal development. More cell death symptoms in the mesophyll cell were observed in the susceptible genotype Sonalika. Relative decrease of HR particularly in Yangmai 6 can be explained by the fact that late penetration attempts failed more common in comparison to early penetration attempts like in Sonalika. While fungus continuously tried to penetrate from newly emerging appressoria during the entire time course of infection.
Candidate genes
The exact function of ABC1 family gene is not known. However, yeast ABC1 suppresses a cytochrome b translation defect and is essential for the electron transfer in the bc 1 complex. Sequence of our barley EST clone was showing similarity to the C-terminal end of Arabidopsis ABC1 line but was located away from ABC domain (250-300 bp) of Arabidopsis. It is suggested that members of ABC1 family are novel chaperones and unrelated to the ABC transporter proteins. The ABC1 family gene was observed down regulated at 8, 24 and 48 h after infection in Bs treated plants. Results were in close proximity with barley results based on MLO/mlo treated with B. sorokiniana.
Role of RNA binding protein Rp 120 is supposed to be associated with cytoskeleton as indicated by its sedimentation behaviour in sucrose density gradient and play a very active role during seed development (Sami et al. 2001). Most of ESTs in TIGR wheat library are found in developing seed or root libraries. However, presences of leaf ESTs in libraries (wlm 4) indicate a role of RNA binding protein Rp 120 in the later growth phase of plant. Down regulation of this cytoskeleton-associated clone (GBN002P06) at 48 and 72 h after inoculation in susceptible genotype, Sonalika indicate that B. sorokiniana may alter or disrupt the cytoskeleton. However, slight up-regulation at 48 and 72 hai by resistant genotype Yangmai 6 may be taken as regaining of RNA binding protein Rp 120 function.
GBN004C05 encoding for Mg-chelatase subunit XANTHA-F observed to be down regulated at 8 h after inoculation in both the varieties indicates that function of the gene is not linked with resistance reaction. Mg-chelatase, a multicomponent enzyme has a very crucial role in the chlorophyll synthesis pathway, owing to its position at the branch point between haem and chlorophyll synthesis. The actual role of Mg-chelatase catalases is the insertion of Mg into protoporphyrin IX. Down regulation may be taken as disrupt supply of Mg or ATPs requirements, since ATPs are required for activation as well as Mg+ chelatation (Lee et al. 1992).
GBN003K10 representing the Actin gene of barley, showed a down regulation at all the time points viz., 8, 24, 48 and 72 h after inoculation compared to control in both the varieties of wheat. Moreover, down regulation was more readable in susceptible genotype Sonalika in comparison to Yangmai 6 (moderate resistant). Actin has a role in the development of cytoskeleton. It can be seen in the light that B. sorokiniana disrupt the basic structure of the cell.
Ferritin gene protects the plant against oxidative damage. Higher Ferritin transcriptional amount absorb more iron particles and protect the plants from the possible damage due to oxidative burst (Maria et al. 1999). B. sorokiniana like other pathogens also cause oxidative damage in the plants (Kumar et al. 2001). GBN003B06, clone for ferritin gene, showed a down regulation at 24 h and a up regulation at 72 h after inoculation with Bs culture in both varieties indicate that pathogen causes initial oxidative damage in infected plant cells but then plant retrieve in the later growth phase of infection and try to overcome these oxidative damages. More up regulation of Ferritin at 72 hai in susceptible genotype might be seen in the light of more damages in susceptible genotype.
Study of B. sorokiniana at cytological level give us an initial idea to develop better understanding about different cellular barriers, play an important role in resistance mechanism. Moreover, knowledge about expression analysis of different candidate genes at Macro array and/or RT-PCR level can be used to improve the present status of disease resistance against B. sorokiniana by developing transgenic plants.
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