Identification and Characterization of Plasmodiophora brassicae Primary Infection Effector Candidates that Suppress or Induce Cell Death in Host and Nonhost Plants.

Clubroot caused by Plasmodiophora brassicaeis one of the most important diseases in cruciferous crops. The recognition of P. brassicae by host plants is thought to occur at the primary infection stage, but the underlying mechanism remains unclear. Secretory proteins as effector candidates play critical roles in the recognition of pathogens and the interactions between pathogens and hosts. In this study, 33 P. brassicae secretory proteins expressed during primary infection were identified through transcriptome, secretory protein prediction, and yeast signal sequence trap analyses. Furthermore, the proteins that could suppress or induce cell death were screened through an Agrobacterium-mediated plant virus transient expression system and a protoplast transient expression system. Two secretory proteins, PBCN_002550 and PBCN_005499, were found to be capable of inducing cell death associated with H2O2 accumulation and electrolyte leakage in Nicotiana benthamiana. Moreover, PBCN_002550 could also induce cell death in Chinese cabbage. In addition, 24 of the remaining 31 tested secretory proteins could suppress mouse Bcl-2-associated X protein-induced cell death, and 28 proteins could suppress PBCN_002550-induced cell death.

Host Range of Plasmodiophora brassicae on Cruciferous Crops and Weeds in China.

Clubroot caused by Plasmodiophora brassicae is an increasingly important soilborne disease in China. The host range of P. brassicae was investigated with 30 cruciferous plants, including 16 crop species, 9 ornamentals, and 5 weeds in field and pot-cultured conditions. In the field, 17 species from five genera produced visible galls, and these included radish, Capsella bursa-pastoris, Orychophragmus violaceus, Sinapis alba, and 13 Brassica crops. In pot-cultured conditions, an additional 13 plant species (11 genera) were determined to be hosts of P. brassicae. Five common weeds were found to be hosts of P. brassicae, including C. bursa-pastoris, Lepidium apetalum, Descurainia sophia, S. alba, and Thellungiella salsuginea. The infection of these plants was confirmed via polymerase chain reaction (PCR) with primers specific to P. brassicae. No galls were found on Matthiola incana roots in the field or in pots and no resting spores of P. brassicae were observed in M. incana roots, although P. brassicae was detected in M. incana roots via PCR. Microscopic examination revealed infection only in the root hairs of M. incana roots. These results suggested that M. incana was highly resistant to P. brassicae in China and could be developed as a bait crop. In total, 297 accessions of oilseed rape were tested in the field, and 3 accessions of Brassica napus and 1 accession of B. juncea were found to be highly resistant to clubroot disease. These resistant resources provide options for managing clubroot in P. brassicae-infested fields.

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1.

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth-associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant-pathogenic protozoan Plasmodiophora brassicae. Through a yeast two-hybrid assay, glutathione S-transferase pull-down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1-regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1-regulated genes in heterogeneous PBZF1-expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae-specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant-pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.

Biological and molecular characterization of a crucifer Tobamovirus infecting oilseed rape.

In China, the tobamovirus that infects oilseed rape has been misdiagnosed as Tobacco mosaic virus (TMV) based on its morphological similarity and serological relatedness. Recently, a tobamovirus has been isolated from oilseed rape in China, which we named Youcai mosaic virus Br (YoMV-Br), according to its biological and molecular characteristics. It had strong infectivity to Cruciferae but less to Solanaceae, Leguminosae, and Cucurbitaceae, and its virion morphology was consistent with that of the tobamoviruses. At high concentrations, it serologically cross reacted with TMV antiserum. The 3' terminal sequence (2,283 nucleotides) of YoMV-Br was determined, including the 3' noncoding region, the CP and MP genes, and the C-terminal part of the replicase gene. Between the MP and CP genes, 77 nucleotides overlapped. Compared with homologous regions of 21 recognized species of Tobamovirus, YoMV-Br had a much higher identity to crucifer species than to other tobamoviruses. Phylogenetic analysis demonstrated that YoMV-Br was closely related to the YoMV cluster of tobamoviruses and distantly to TMV, so that they likely belong to different strains of the same species.

Jasmonic Acid-Mediated Aliphatic Glucosinolate Metabolism Is Involved in Clubroot Disease Development in Brassica napus L.

Glucosinolate (GSL) is associated with clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae. Due to the complicated composition of GSLs, their exact role in clubroot disease development remains unclear. By investigating clubroot disease resistance in cruciferous plants and characterizing the GSL content in seeds, we can determine if clubroot disease development is related to the components of GSLs. The difference in the infection process between Matthiola incana L. (resistant) and Brassica napus L. (susceptible) was determined. Root hair infection was definitely observed in both resistant and susceptible hosts, but no infection was observed during the cortical infection stage in resistant roots; this finding was verified by molecular detection of P. brassicae via PCR amplification at various times after inoculation. Based on the time course detection of the contents and compositions of GSLs after P. brassicae inoculation, susceptible roots exhibited increased accumulation of aliphatic, indolic, and aromatic GSLs in B. napus, but only aromatic GSLs were significantly increased in M. incana. Gluconapin, which was the main aliphatic GSL in B. napus and present only in B. napus, was significantly increased during the secondary infection stage. Quantification of the internal jasmonic acid (JA) concentration showed that both resistant and susceptible plants exhibited an enhanced level of JA, particularly in susceptible roots. The exogenous JA treatment induced aliphatic GSLs in B. napus and aromatic GSLs in M. incana. JA-induced aromatic GSLs may be involved in the defense against P. brassicae, whereas aliphatic GSLs induced by JA in B. napus likely play a role during the secondary infection stage. Three candidate MYB28 genes regulate the content of aliphatic GSLs identified in B. napus; one such gene was BnMYB28.1, which was significantly increased following both the treatment with exogenous JA and P. brassicae inoculation. In summary, the increased content of JA during the secondary infection stage may induce the expression of BnMYB28.1, which caused the accumulation of aliphatic GSLs in clubroot disease development.

Differentiation of Peanut Seedborne Potyviruses and Cucumoviruses by RT-PCR.

Seedborne peanut viruses pose important constraints to peanut production and safe movement of germ plasm. They also pose a risk of accidental introduction into previously disease-free regions. We have developed reverse transcription-polymerase chain reaction (RT-PCR) assays based on identical cycling parameters which identified peanut stripe, Peanut mottle, Peanut stunt, and Cucumber mosaic viruses through production of specific DNA fragments of 234 bp, 327 bp, 390 bp, and 133 bp, respectively. Assay sensitivity in the picogram range was achieved. The two potyviruses and two cucumoviruses could be differentiated using duplex RT-PCR assays. These assays should be useful for testing peanut leaves or seeds for virus identification in epidemiological studies, seed testing or in post-entry quarantine.