Brassicae Infection Research Articles

Identification of Expressed Genes During Infection of Chinese Cabbage (Brassica rapa subsp. pekinensis) by Plasmodiophora brassicae

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.

Proteome-level changes in the roots of Brassica napus as a result of Plasmodiophora brassicae infection

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, has emerged as an important new disease of canola ( Brassica napus) in Alberta, Canada. Changes in the root protein profile were examined by two-dimensional gel electrophoresis at 12, 24, 48 and 72 h after inoculation of a susceptible canola genotype with the pathogen. A total of 20 protein spots were identified as either increasing (13 spots) or decreasing (7 spots) in intensity relative to water-treated controls. The identities of these proteins were established by MS/MS, and included proteins involved in lignin biosynthesis, cytokinin metabolism, glycolysis, intracellular calcium homeostasis, and the detoxification of ROS. Decreased abundance of adenosine kinase, which is involved in cytokinin homeostasis, supported previous reports indicating a key role for cytokinin in the early phases of clubroot infection. An approximately sixfold reduction in caffeoyl-CoA O-methyltransferase abundance suggested a reduction in host lignin biosynthesis after pathogen challenge, and is consistent with the compatible nature of the interaction examined. Interestingly, levels of enzymes involved in ROS metabolism declined sharply at 12 h after inoculation, but increased at 24–72 h. Collectively, these data suggest major changes in canola metabolism shortly after challenge by the pathogen, which may result in the susceptibility of the host.

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.

Increase in Indole-3-acetic Acid(IAA) level and Nitrilase Activity in Turnips Induced by Plasmodiophora brassicae Infection.

Indole acetic acid (IAA) level and nitrilase activity were measured in infected and healthy turnips, Brassica campestris L., subsp. Rapa, over a time course to confirm increases in IAA levels upon infection by Plasmodiophora brassicae and to investigate underlying mechanisms. Healthy and infected seedlings were assayed from 20 to 90 days after sowing. IAA levels in both roots fluctuated similarly over days 20-35. By day 45, IAA in infected roots increased to five-fold over healthy roots then decreased to the level of healthy roots by day 90. Nitrilase activity was negligible on days 20 and 25 but increased thereafter in infected and healthy roots. However, activity in healthy roots decreased substantially by day 40 while infected roots showed a continued increase to day 45 then decreased to a low level. These findings suggest that IAA concentration increases after P. brassicae infection, possibly due to IAA synthesis via pathways involving nitrilase.

Isolation and characterization of novel defence‐related genes induced by copper, salicylic acid, methyl jasmonate, abscisic acid and pathogen infection in Brassica carinata

Summary To examine the defence response in Brassica carinata we differentially screened a cDNA library made from CuCl(2)-treated (Cu) leaves. The sequence of 17 of the 27 cDNA clones examined that showed Cu-induction had a high similarity to defence genes from other plant species. Among other clones that showed higher expression in the Cu leaves were two cDNAs encoding polypeptides of 351 and 250 amino acids, designated BcCJS1 and BcCJAS1. BcCJS1 had similarity to S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase from Clarkia breweri. However, the enzyme activity was not found in extracts from E. coli expressing BcCJS1. BcCJAS1 did not show extensive similarity to any genes with known function in the databases but it did contain three regions of amino acid sequence that are frequently found in amidotransferases. A third Cu-induced mRNA, Bcp6PGL, showed very high (86%) similarity to a putative 6-phosphogluconolactonase (6PGL) from Arabidopsis thaliana. In addition to Cu induction, BcCJS1 expression was induced by methyl jasmonate (MeJA) and salicylic acid (SA), BcCJAS1 expression by MeJA, SA and abscisic acid and Bcp6PGL expression by MeJA. The expression of all three genes increased after Alternaria brassicae infection. BcCJS1 and BcCJAS1 were induced within 1 h after MeJA- but not until 3 h after SA-treatment. The expression of both genes was systemically induced after infection with a compatible or incompatible fungal pathogen. SA systemically induced only BcCJAS1. The effects of various inhibitors of signalling pathways on expression of the three genes were studied.

Plasmodiophora brassicae -induced expression of pyk20, an Arabidopsis thaliana gene with glutamine-rich domain

The expression of the pyk20 gene of Arabidopsis thaliana, which encodes a protein containing a glutamine-rich domain is up-regulated during Plasmodiophora brassicae infection. Transgenic A. thaliana plants harbouring a β-glucuronidase (uid A) reporter gene under the control of the pyk20 gene promoter were grown in soil and infected with P. brassicae resting spores. GUS expression in non-infected plants was found in stipules, apical meristem, leaf vascular tissues, vascular tissue of roots and in the root tips. After infection with P. brassicae, GUS staining was observed in the root hairs during primary infection and in galls in roots and hypocotyls during secondary infection phase. GUS expression during primary infection was also detected in a small number (approx. 0.01%) of zoosporangia. Sections of the GUS-stained galls showed reporter gene expression in infected and non-infected tissues. Northern-blot analysis using a pyk20 cDNA clone as a probe confirmed responsiveness of the pyk20 gene to P. brassicae infection.

Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis.

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development is discussed.

179 Control of Clubroot on Chinese Mustard and Cauliflower using Meadowfoam (Limnanthes alba) Seedmeal or Screenings

Seedmeal (MSM) and screenings (MS) of meadowfoam (Limnanthes alba H.) were evaluated for their influence on the development of clubroot caused by Plasmodiophora brassicae in potted seedlings. Treatments included MSM at 0%, 5%, 10%, and 20% (by vol.); MSM at 10% (by vol.) plus an 8 oz. application of 3% H2O2 per pot; and MS at 10% (by vol.) pre-sowing incorporation into potting media (Sunshine Mix #1) with a 10% (by vol.) clay-loam soil known to be infested with P. brassicae resting spores. One-hundred percent Sunshine mix #1 was used as a control medium. Following media preparation, seeds of Chinese mustard (Brassica chinensis) and cauliflower (Brassica oleracea var. botrytis `Snowball Y Improved') were sown every 7 days for 4 weeks. Symptoms of P. brassicae infection (clubbing or rotting of roots) occurred in 70% to 90% of all plants grown in pots with media containing infested soil and no MSM or MS, with disease severity ranging from <25% to >50% of root systems clubbed or rotted. Chinese mustard seedlings had more clubbing compared to cauliflower seedlings. All plants grown in media containing MSM or MS showed no clubbing or rotting. Plants grown in 20% MSM or 10% MSM plus a 3% H2O2 had symptoms of phytotoxicity. Plants grown in 10% MSM or 10% MS were taller compared to controls. Although plants grown in MSM and MS showed no clubroot symptoms, asymptotic presence of pathogen has not been excluded.

The host range of Plasmodiophora brassicae and its relationship to endogenous glucosinolate content

The host range of the soilborne obligate biotroph, Plasmodiophora brassicae was investigated. Evidence is presented that infection by P. brassicae might occur in non‐Brassica species, leading to the potential formation of resting spores. Structures resembling P. brassicae were found in the root cortex of Tropaeolum majus, Carica papaya, Reseda alba and Beta vulgaris as demonstrated by scanning electron microscopy. Inoculation of Brassica rapa roots with spores extracted from either T. majus or B. vulgaris roots which had been previously inoculated with P. brassicae led to development of clubroot in the roots of B. rapa. It was also shown that the development of the symptom might be correlated with glucosinolate content, although other host factors are implicated in the B. vulgaris interaction with P. brassicae. In the glucosinolate‐containing non‐Brassicas, T. majus and C. papaya, the concentrations of benzylglucosinolate increased markedly in roots inoculated with P. brassicae, compared with the controls. There were also increases in concentrations of benzylglucosinolate in leaves of T. majus after P. brassicae infection. However, in R. alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls. High root concentrations of 2‐OH‐2‐phenylethylglucosinolate (glucobarbarin) compared with low root indole glucosinolates in this species might limit P. brassicae infection and development. The importance of our investigations in relation to cultivation of non‐Brassica species on fields infested with P. brassicae is discussed.