Recognition of avirulence gene AvrLm1 from hemibiotrophic ascomycete Leptosphaeria maculans triggers salicylic acid and ethylene signaling in Brassica napus

Interaction of a plant with a fungal pathogen is an encounter with hundreds of molecules. In contrast to this, a single molecule often decides between the disease and resistance. In the present article, we describe the defense responses triggered by AvrLm1, an avirulence gene from a hemibiotrophic ascomycete, Leptosphaeria maculans, responsible for an incompatible interaction with Brassica napus. Using multiple hormone quantification and expression analysis of defense-related genes, we investigated signaling events in Rlm1 plants infected with two sister isolates of L. maculans differentiated by the presence or absence of AvrLm1. Infection with the isolate carrying AvrLm1 increased the biosynthesis of salicylic acid (SA) and induced expression of the SA-associated genes ICS1, WRKY70, and PR-1, a feature characteristic of responses to biotrophic pathogens and resistance gene-mediated resistance. In addition to SA-signaling elements, we also observed the induction of ASC2a, HEL, and CHI genes associated with ethylene (ET) signaling. Pharmacological experiments confirmed the positive roles of SA and ET in mediating resistance to L. maculans. The unusual cooperation of SA and ET signaling might be a response to the hemibiotrophic nature of L. maculans. Our results also demonstrate the profound difference between the natural host B. napus and the model plant Arabidopsis in their response to L. maculans infection.     

Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris.

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus 'Surpass 400' are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. 'Surpass 400' also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of 'Surpass 400' was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on 'Surpass 400' was different from those observed on AD9 and MC1-8, indicating that 'Surpass 400' carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of 'Surpass 400' to most of the isolates tested were indistinguishable from those of AD49, which suggested 'Surpass 400' might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in 'Surpass 400'. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants

Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a 'gene for gene' manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans.     

Pathways of infection of Brassica napus roots by Leptosphaeria maculans

Infection of Brassica napus cotyledons and leaves by germinating ascospores of Leptosphaeria maculans leads to production of leaf lesions followed by stem cankers (blackleg). Leptosphaeria maculans also causes root rot but the pathway of infection has not been described. An L. maculans isolate expressing green fluorescent protein (GFP) was applied to the petiole of B. napus plants. Hyphal growth was followed by fluorescence microscopy and by culturing of sections of plant tissue on growth media. Leptosphaeria maculans grew within stem and hypocotyl tissue during the vegetative stages of plant growth, and proliferated into the roots within xylem vessels at the onset of flowering. Hyphae grew in all tissues in the stem and hypocotyl, but were restricted mainly to xylem tissue in the root. Leptosphaeria maculans also infected intact roots when inoculum was applied directly to them and hyphae entered at sites of lateral root emergence. Hyphal entry may occur at other sites but the mechanism is uncertain as penetration structures were not observed. Infection of B. napus roots by L. maculans can occur via above- and below-ground sources of inoculum, but the relative importance of the infection pathways under field conditions is unknown.     

Enhanced pathogenicity of Leptosphaeria maculans Pycnidiospores from paired co-inoculation of Brassica napus cotyledons with ascospores

BACKGROUND AND AIMS: Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) worldwide, including Australia. In most cases, the severity of the disease in the field is related to infections caused by airborne ascospores. In contrast, pycnidiospores originating from leaf and stem lesions and stubble are widely assumed to play only a relatively minor role in the epidemiology of blackleg. It is not clear whether, under certain conditions, pycnidiospores can cause severe disease in the field. The aim of the work reported was to determine if the pathogenicity of pycnidiospores is enhanced by paired co-inoculation of B. napus cotyledons with ascospores. METHODS: Three investigations were carried out under controlled-environment conditions using various L. maculans isolates and B. napus cultivars with different levels of host resistance to blackleg. KEY RESULTS: In all three experiments, co-inoculation with ascospores increased the ability of pycnidiospores to cause more disease on B. napus than when inoculations consisted of pycnidiospores alone. This effect was significantly influenced by the host resistance of the cultivar, but overall was independent of the L. maculans isolate used in the different experiments. This effect was also independent of timing of inoculation with the ascospores, with increased disease from pycnidiospores occurring on the cotyledon of the seedling in situations where inoculations with ascospores were carried out 0, 1 or 2 d after pycnidiospore inoculation. This enhanced pathogenicity of pycnidiospores was evident even when low concentrations of pycnidiospores were applied to the other cotyledon of the same seedling. CONCLUSIONS: These results may explain continuing severe blackleg disease cycles throughout the cropping season even when ascospore fallout was low or constrained only to a brief period or phase of the cropping season, and suggest that disease epidemics may be polycyclic rather than monocyclic.     

Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans

Restriction enzyme mediated insertional mutagenesis using a plasmid, pUCATPH, that confers hygromycin resistance, generated loss-of-pathogenicity mutants of Leptosphaeria maculans, the fungus that causes blackleg disease of Brassica napus. Of 516 L. maculans transformants analysed, 12 were pathogenicity mutants. When eight of these mutants were crossed to an isolate that attacks B. napus, cosegregation of pUCATPH sequences and loss of pathogenicity was not observed, suggesting that these mutations were not linked to plasmid sequences. In seven of eight crosses analysed, progeny with the hygromycin resistance gene were hygromycin-sensitive. Sequence analysis of an amplified fragment of pUCATPH in six clones derived from one 'silenced' progeny showed mutation of GC to AT on one DNA strand, reminiscent of repeat-induced point mutation (RIP) in Neurospora crassa. One loss-of-pathogenicity mutant had pUCATPH inserted in the promoter of a gene with an open reading frame of 529 amino acids that had no database match. Reintroduction of a wild-type copy of the gene to this mutant restored the ability to form lesions on cotyledons of B. napus.     

Regulation of Proteolytic Enzyme Activity in Lactococcus lactis

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.     

B-genome derived resistance to Leptosphaeria maculans in near isogenic Brassica napus lines is independent of glucosinolate profile

The role of the glucosinolate-myrosinase system in resistance to Leptosphaeria maculans was studied by monitoring changes in glucosinolate profiles in leaf tissue surrounding the site of inoculation. Susceptible Brassica napus cv. Hanna, resistant B. nigra and near isogenic lines derived from interspecific hybrids between the two species were compared. Expression of myrosinase binding protein and presence of genetic markers were also assayed. No correlation between degree of resistance and amount of sinigrin or other aliphatic glucosinolates was found. However, in time course experiments the glucosinolate profile of the L. maculans inoculated plants differed significantly from the water-inoculated control plants in the amount of 4-methoxy-glucobrassicin observed. Five to eight days post-inoculation an increased level of 4-methoxy glucobrassicin, ranging from 30% to 47% on average, was found in the inoculated plants, whilst controls varied between 7.6% and 9.2%. This increase was seen both in susceptible and resistant material. Other changes observed could mainly be assigned as effects of wounding. Although inoculation with L. maculans elicited changes in the leaf indolyl glucosinolate profiles, in the material studied, these changes could not be correlated to resistance against the fungus.     

Antagonism of Erwinia herbicola towards Leptosphaeria maculans causing blackleg disease of Brassica napus

Phyllosphere micro-organisms of Brassica napus were isolated and their antagonism against Leptosphaeria maculans , causal agent of blackleg disease, was tested in vitro . In paired culture, Erwinia herbicola was found to be highly antagonistic to L. maculans. Bioassay of the culture filtrate of the bacterium against the test fungus revealed that Erw. herbicola secretes an antifungal substance into the culture medium. This substance was partially thermolabile and markedly reduced the germination and germ tube length of L. maculans . Aqueous bacterial suspensions and cold-sterilized culture filtrates, when applied to the seedlings prior to inoculation, significantly reduced the severity of blackleg disease.     

The effect of specific inducers on the biosynthesis of lytic enzymes by a strain of Streptomyces recifensis var. lyticus

The effect of specific inductors at different stages of Streptomyces recifensis var. lyticus 2435 cultivation was studied. It is shown that introduction of killed bacterial cells into inoculum not only influences the level of accumulation of lytic enzymes by the strain S. recifensis var. lyticus 2435 but also determines the qualitative composition of the synthesized complex. The yield of bacteriological glycosidases grows with introduction of the Micrococcus lysodeikticus cells into the inoculum or of dissolvable chitin into the enzymic medium. The possibility to preserve the initial level of the lytic activity in the producer during re-inoculations by introducing the Bacillus subtilis cells into the cultivation medium has been studied.     

Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.     

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.     

Potato virus Y NIa protease activity is not sufficient for elicitation of Ry-mediated disease resistance in potato

Ry confers extreme resistance (ER) to all strains of potato virus Y (PVY). In previous work, we have shown that the protease domain of the nuclear inclusion a protease (NIaPro) from PVY is the elicitor of the Ry-mediated resistance and that integrity of the protease active site is required for the elicitation of the resistance response. Two possibilities arise from these results: first, the structure of the active protease has elicitor activity; second, NIa-mediated proteolysis is required to elicit the resistance response. To resolve these possibilities, the NIaPro from PVY was randomly mutagenised and the clones obtained were screened for elicitation of cell death as an indicator of resistance and proteolytic activity. We did not find any mutants that had retained the ability to elicit cell death but had lost protease activity, as measured by processing of the NIa cleavage site in the viral genome. This was consistent with the idea that protease activity is necessary for elicitor activity. However, protease activity was not sufficient because we found three elicitor-defective mutants in which there was a high level of protease activity in this assay.     

真菌诱导子对悬浮培养南方红豆杉细胞态势及紫杉醇合成的影响

在南方红豆杉细胞悬浮培养过程中,研究了在指数生长期的末期加入真菌诱导子(尖孢镰刀菌的胞壁组分粗提物)对细胞态势及紫杉醇合成的影响。结果表明,真菌诱导子能在短期内激发细胞的防御性应答反应而产生氧迸发,细胞的氧化还原态势发生了明显的变化,培养基发生碱化现象,表征酶含量的蛋白质含量明显提高,SOD、POD、CAT的活力出现波动性变化,并具有一定的时序性。同时,南方红豆杉悬浮培养体系中产次生代谢途径中重要的酶PAL的活力得到提高,紫杉醇的合成被加强,产量得到了显著提高,达到了对照组的5倍左右。