Leptosphaeria maculans effector AvrLm4-7 affects salicylic acid (SA) and ethylene (ET) signalling and hydrogen peroxide (H2 O2 ) accumulation in Brassica napus.

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans.

Genome-wide transcriptomic analyses provide insights into the lifestyle transition and effector repertoire of Leptosphaeria maculans during the colonization of Brassica napus seedlings.

Molecular interaction between the causal agent of blackleg disease, Leptosphaeria maculans (Lm), and its host, Brassica napus, is largely unknown. We applied a deep RNA-sequencing approach to gain insight into the pathogenicity mechanisms of Lm and the defence response of B. napus. RNA from the infected susceptible B. napus cultivar Topas DH16516, sampled at 2-day intervals (0-8 days), was sequenced and used for gene expression profiling. Patterns of gene expression regulation in B. napus showed multifaceted defence responses evident by the differential expression of genes encoding the pattern recognition receptor CERK1 (chitin elicitor receptor kinase 1), receptor like proteins and WRKY transcription factors. The up-regulation of genes related to salicylic acid and jasmonic acid at the initial and late stages of infection, respectively, provided evidence for the biotrophic and necrotrophic life stages of Lm during the infection of B. napus cotyledons. Lm transition from biotrophy to necrotropy was also supported by the expression function of Lm necrosis and ethylene-inducing (Nep-1)-like peptide. Genes encoding polyketide synthases and non-ribosomal peptide synthetases, with potential roles in pathogenicity, were up-regulated at 6-8 days after inoculation. Among other plant defence-related genes differentially regulated in response to Lm infection were genes involved in the reinforcement of the cell wall and the production of glucosinolates. Dual RNA-sequencing allowed us to define the Lm candidate effectors expressed during the infection of B. napus. Several candidate effectors suppressed Bax-induced cell death when transiently expressed in Nicotiana benthamaina leaves.

Comparison of Soybean Transformation Efficiency and Plant Factors Affecting Transformation during the Agrobacterium Infection Process.

The susceptibility of soybean genotype to Agrobacterium infection is a key factor for the high level of genetic transformation efficiency. The objective of this study is to evaluate the plant factors related to transformation in cotyledonary nodes during the Agrobacterium infection process. This study selected three genotypes (Williams 82, Shennong 9 and Bert) with high transformation efficiency, which presented better susceptibility to Agrobacterium infection, and three low transformation efficiency genotypes (General, Liaodou 16 and Kottman), which showed a relatively weak susceptibility. Gibberellin (GA) levels and soybean GA20ox2 and CYP707A2 transcripts of high-efficiency genotypes increased and were higher than those of low-efficiency genotypes; however, the opposite performance was shown in abscisic acid (ABA). Higher zeatin riboside (ZR) content and DNA quantity, and relatively higher expression of soybean IPT5, CYCD3 and CYCA3 were obtained in high-efficiency genotypes. High-efficiency genotypes had low methyl jasmonate (MeJA) content, polyphenol oxidase (PPO) and peroxidase (POD) activity, and relatively lower expression of soybean OPR3, PPO1 and PRX71. GA and ZR were positive plant factors for Agrobacterium-mediated soybean transformation by facilitating germination and growth, and increasing the number of cells in DNA synthesis cycle, respectively; MeJA, PPO, POD and ABA were negative plant factors by inducing defence reactions and repressing germination and growth, respectively.

Ras GTPase activating protein CoIra1 is involved in infection-related morphogenesis by regulating cAMP and MAPK signaling pathways through CoRas2 in Colletotrichum orbiculare.

Colletotrichum orbiculare is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP-PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1-Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP-CoRAS2. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2.

The KEEP ON GOING protein of Arabidopsis regulates intracellular protein trafficking and is degraded during fungal infection.

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.

Mungbean plants expressing BjNPR1 exhibit enhanced resistance against the seedling rot pathogen, Rhizoctonia solani.

Mungbean, Vigna radiata (L.) Wilczek is an important pulse crop that is widely cultivated in semi- arid tropics. The crop is attacked by various soil-borne pathogens like Rhizoctonia solani, which causes dry rot disease and seriously affects its productivity. Earlier we characterized the non-expressor of pathogenesis related gene-1(BjNPR1) of mustard, Brassica juncea, the counterpart of AtNPR1 of Arabidopsis thaliana. Here, we transformed mungbean with BjNPR1 via Agrobacterium tumefaciens. Because of the recalcitrant nature of mungbean, the effect of some factors like Agrobacterium tumefaciens strains (GV2260 and LBA4404), pH, L: -cysteine and tobacco leaf extract was tested in transformation. The transgenic status of 15 plants was confirmed by PCR using primers for nptII. The independent integration of T-DNA in transgenic plants was analyzed by Southern hybridization with an nptII probe and the expression of BjNPR1 was confirmed by RT-PCR. Some of the T(0) plants were selected for detached leaf anti-fungal bioassay using the fungus Rhizoctonia solani, which showed moderate to high level of resistance depending on the level of expression of BjNPR1. The seedling bioassay of transgenic T(2) plants indicated resistance against dry rot disease caused by R. solani.

The tomato UV-damaged DNA-binding protein-1 (DDB1) is implicated in pathogenesis-related (PR) gene expression and resistance to Agrobacterium tumefaciens.

Plants defend themselves against potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs). However, the molecular mechanisms underlying this PAMP-triggered immunity (PTI) are largely unknown. In this study, we show that tomato HP1/DDB1, coding for a key component of the CUL4-based ubiquitin E3 ligase complex, is required for resistance to Agrobacterium tumefaciens. We found that the DDB1-deficient mutant (high pigment-1, hp1) is susceptible to nontumorigenic A. tumefaciens. The efficiency of callus generation from the hp1 cotyledons was extremely low as a result of the necrosis caused by Agrobacterium infection. On infiltration of nontumorigenic A. tumefaciens into leaves, the hp1 mutant moderately supported Agrobacterium growth and developed disease symptoms, but the expression of the pathogenesis-related gene SlPR1a1 and several PTI marker genes was compromised at different levels. Moreover, exogenous application of salicylic acid (SA) triggered SlPR1a1 gene expression and enhanced resistance to A. tumefaciens in wild-type tomato plants, whereas these SA-regulated defence responses were abolished in hp1 mutant plants. Thus, HP1/DDB1 may function through interaction with the SA-regulated PTI pathway in resistance against Agrobacterium infection.

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition.

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.

Indução de fitoalexinas em soja e sorgo por preparações de Saccharomyces boulardii / Induction of phytoalexins in soybean and sorghum by Saccharomyces boulardii

O gênero Saccharomyces tem sido usado como indutor de resistência ou para controle biológico em muitos patossistemas. Neste trabalho objetivou-se a indução de fitoalexinas em mesocótilos de sorgo e cotilédones de soja pela levedura Saccharomyces boulardii na forma do produto comercial Floratil (Merck) (com 2 x 106 células/mg produto comercial - pc) e massa de células obtidas de meio líquido YEPG (primeiramente com 14 dias de cultivo e, posteriormente, com 7, 14, 21, 28 e 35 dias) ambos em concentrações de 0,005; 0,05; 0,5; 5; 15 e 25 mg/mL, além de filtrado desse meio nas concentrações de 0,01; 0,1; 1; 5; 10 e 20%. Como tratamentos controle utilizou-se água e S. cerevisiae (25 mg/mL de pc) para soja e água e acibenzolar-S-metil (ASM) (125 mg i.a./L) para sorgo. Em soja os três produtos apresentaram efeito dose-dependente, com ajustes de equações de 1° grau e R2 de 0,64; 0,94 e 0,98 não tendo efeito do tempo de cultivo da levedura na indução de fitoalexinas. Em sorgo apenas o filtrado e Floratil tiveram efeito dose-dependente com equação de 1° grau e R2 de 0,63 e 0,94 respectivamente e obteve-se nos diferentes dias de cultivo R2 de 0,62 com a massa de células somente. Portanto, pode-se evidenciar o potencial indutor de fitoalexinas dos produtos a base de S. boulardii para ensaios com indução de resistência em patossistemas envolvendo sorgo e soja.

Saccharomyces yeast compounds have been used as a resistance elicitor or for biological control in many pathosystems. Thus, the aim of this research was to verify the induction of phytoalexins in sorghum mesocotyls and soybean cotyledons by using Saccharomyces boulardii in the form of the commercial product Floratil (Merck) (with 2 x 106 cells/mg) and yeast-cell mass obtained from liquid culture in YEPG medium (with 7, 14, 21, 28 and 35 days old), both at concentrations of 0.005, 0.05, 0.5, 5, 15 and 25 mg/mL, as well as the filtrate of this medium in concentrations of 0.01, 0.1, 1, 5, 10 and 20%. The control treatments consisted of distilled water and S. cerevisiae (25 mg of commercial product per mL) for the soybean tests and distilled water and acibenzolar-S-methyl (125 mg of active ingredient per L) for the sorghum tests. In soybeans the three tested S. boulardii products presented a dose-dependent effect with R2 of 0.64, 0.94 and 0.98 for the culture filtrate, cell suspension and commercial product of S. boulardii, respectively, with no effect of culture time of yeasts on phytoalexin induction. In sorghum, only the culture filtrate and Floratil presented a dose-dependent effect, with R2 of 0.63 and 0.94, respectively, and the cell suspension of S. boulardii showed dependence of culture time with R2 of 0.62. Thus, S. boulardii and its derivates induce phytoalexins and have potential to be used as an elicitor for assays with induction resistance in pathosystems involving sorghum and soybean plants.

Fusarium graminearum exploits ethylene signalling to colonize dicotyledonous and monocotyledonous plants.

Ethylene signalling affects the resistance of dicotyledonous plant species to diverse pathogens but almost nothing is known about the role of this pathway in monocotyledonous crop species. Fusarium graminearum causes Fusarium head blight (FHB) of cereals, contaminating grain with mycotoxins such as deoxynivalenol (DON). Very little is known about the mechanisms of resistance/susceptibility to this disease. Genetic and chemical genetic studies were used to examine the influence of ethylene (ET) signalling and perception on infection of dicotyledonous (Arabidopsis) and monocotyledonous (wheat and barley) species by F. graminearum. Arabidopsis mutants with reduced ET signalling or perception were more resistant to F. graminearum than wild-type, while mutants with enhanced ET production were more susceptible. These findings were confirmed by chemical genetic studies of Arabidopsis, wheat and barley. Attenuation of expression of EIN2 in wheat, a gene encoding a core component of ethylene signalling, reduced both disease symptoms and DON contamination of grain. Fusarium graminearum appears to exploit ethylene signalling in both monocotyledonous and dicotyledonous species. This demonstration of translation from model to crop species provides a foundation for improving resistance of cereal crops to FHB through identification of allelic variation for components of the ethylene-signalling pathway.

Molecular interaction between Methylobacterium extorquens and seedlings: growth promotion, methanol consumption, and localization of the methanol emission site.

Four Methylobacterium extorquens strains were isolated from strawberry (Fragaria x ananassa cv. Elsanta) leaves, and one strain, called ME4, was tested for its ability to promote the growth of various plant seedlings. Seedling weight and shoot length of Nicotiana tabacum, Lycopersicon esculentum, Sinapis alba, and Fragaria vesca increased significantly in the presence of the pink-pigmented facultative methylotroph (PPFM), but the germination behaviour of seeds from six other plants was not affected. The cell-free supernatant of the bacterial culture stimulated germination, suggesting the production of a growth-promoting agent by the methylotroph. Methanol emitted from N. tabacum seedlings, as determined by proton-transfer-reaction mass spectrometry (PTR-MS), ranged from 0.4 to 0.7 ppbv (parts per billion by volume), while significantly lower levels (0.005 to 0.01 ppbv) of the volatile alcohol were measured when the seedlings were co-cultivated with M. extorquens ME4, demonstrating the consumption of the gaseous methanol by the bacteria. Additionally, by using cells of the methylotrophic yeast Pichia pastoris transformed with the pPICHS/GFP vector harbouring a methanol-sensitive promoter in combination with the green fluorescence protein (GFP) reporter gene, stomata were identified as the main source of the methanol emission on tobacco cotyledons. Methylobacterium extorquens strains can nourish themselves using the methanol released by the stomata and release an agent promoting the growth of the seedlings of some crop plants.

Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction.

Lipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium.

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.

Salicylic acid and ethylene pathways are differentially activated in melon cotyledons by active or heat-denatured cellulase from Trichoderma longibrachiatum.

Infiltration of cellulase (EC 3.2.1.4) from Trichoderma longibrachiatum into melon (Cucumis melo) cotyledons induced several key defense mechanisms and hypersensitive reaction-like symptoms. An oxidative burst was observed 3 hours after treatment and was followed by activation of ethylene and salicylic acid (SA) signaling pathways leading to marked induction of peroxidase and chitinase activities. The treatment of cotyledons by heat-denatured cellulase also led to some induction of peroxidase and chitinase activities, but the oxidative burst and SA production were not observed. Co-infiltration of aminoethoxyvinil-glycine (an ethylene inhibitor) with the active cellulase did not affect the high increase of peroxidase and chitinase activities. In contrast, co-infiltration of aminoethoxyvinil-glycine with the denatured enzyme blocked peroxidase and chitinase activities. Our data suggest that the SA pathway (induced by the cellulase activity) and ethylene pathway (induced by heat-denatured and active protein) together coordinate the activation of defense mechanisms. We found a partial interaction between both signaling pathways since SA caused an inhibition of the ethylene production and a decrease in peroxidase activity when co-infiltrated with denatured cellulase. Treatments with active or denatured cellulase caused a reduction in powdery mildew (Sphaerotheca fuliginea) disease.

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

Indole acetic acid and its metabolism in root nodules of a monocotyledonous tree Roystonea regia.

A monocotyledonous tree, Roystonea regia, was found to bear root nodules. The root nodules contained a high amount (16.9 microg/g fresh mass) of indole acetic acid (IAA). A big tryptophan pool (1555.1 microg/g fresh mass) was found in the root nodules, which might serve as a source of IAA production. The presence of IAA-metabolizing enzymes IAA oxidase and peroxidase indicated metabolism of IAA in the root nodules. The symbiont isolated from the root nodules of R. regia, a Rhizobium sp., produced high amount of IAA in culture when supplemented with tryptophan. The possible role of this IAA production in the monocotyledonous tree-Rhizobium symbiosis is discussed.

Defence-related gene activation during an incompatible interaction between Stagonospora (Septoria) nodorum and barley (Hordeum vulgare L.) coleoptile cells.

Two previously unidentified cDNA clones (bsi1 and bpr1-1) were isolated by differential hybridization from a cDNA library of Stagonospora (Septoria) nodorum (Berk) Castellani & E.G. Germano (teleomorph Phaeosphaeria (Leptosphaeria) nodorum (E. Muller) Hedjaroude-challenged barley (Hordeum vulgare L.) coleoptiles. bsi1 encoded a cysteine-rich protein containing 89 amino acids (aa) with a relative molecular mass (M(r)) of 9405. Protein sequence homologies showed that Bsi1 was very similar to an aluminium-induced protein from wheat and indicated that it was related to the Bowman-Birk-type proteinase inhibitors (BB-PIs). The predicted aa sequence of Bsi1 contained an N-terminal secretory signal sequence which implied that the protein was exported. The other clone, bprl-1, which was truncated at the 5' end, encoded a type-1 pathogenesis-related (PR-1) protein. The complete sequence of bpr1-1 was obtained after cloning a barley genomic DNA fragment and was shown to encode a basic protein containing 174 aa with a M(r) of 18 859. The deduced aa sequence of bpr1-1 contained both an N-terminal secretory signal sequence and a charged C-terminal extension. This latter sequence may represent a vacuolar targeting signal. bsil and bpr1-1 and four other defence-related genes (encoding 1,3-beta-glucanase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a homologue of a putative wheat peroxidase, and barley leaf-specific thionin), showed increased transcription levels in S. nodorum-challenged coleoptiles, although their pattern of accumulation varied after inoculation (a.i.). The potential role of these induced genes in defence against fungal attack is discussed.