MicroRNAs in the response of non-heading Chinese cabbage to Hyaloperonospora parasitica (downy mildew) infection

Background MicroRNAs (miRNAs) are a subset of endogenous small non-coding ~21 nucleotide RNAs, which are playing important roles in development, abiotic stress, and pathogen responses. Downy mildew, caused by Hyaloperonospora parasitica , is a severe fungal disease in non-heading Chinese cabbage ( Brassica rapa ssp. chinensis ). However, miRNAs and their targets involved in the response of non-heading Chinese cabbage to downy mildew were still unclear. Results Here, fifteen small RNA libraries were generated from non-heading Chinese cabbage leaves at different time points after H. parasitica inoculation. By deep sequencing, we identified 197 known miRNAs and 112 novel miRNAs. According to data analysis, 8 known miRNAs and 8 novel miRNAs were found to be responsive to downy mildew infection in non-heading Chinese cabbage. Expression levels of 12 predicted miRNAs target genes were determined by reverse transcription-quantitative polymerase chain reaction (qRT-PCR). In this study, we proposed a regulatory network showing that non-heading Chinese cabbage defense response to downy mildew by repressing cell wall pectin, lipid and cellulose catabolic process ( PME12 , PME17 , PLAIVA and GDPDL4 ) and callose deposition ( CYP79B2 ), repressing indole glucosinolate metabolic process ( CYP81F3 ), repressing respiratory burst ( PUB24 ), repressing programmed cell death ( CRK5 ), and inducing defense related gene expression ( ROS1 , LECRK-VII.2 , BAK7 and WRKY48 ). Conclusions This research identified miRNAs associated with downy mildew infection in non-heading Chinese cabbage. Target genes and network analysis contributed to understanding the interaction between plant and pathogen.

Albugo candida (white rust) suppresses resistance to downy mildew pathogens in Arabidopsis thaliana

Arabidopsis thaliana accessions were inoculated with incompatible isolates of downy mildews, following pre-inoculation with compatible Albugo candida. Three isolates of Hyaloperonospora parasitica subsp. A. thaliana, an isolate of H. parasitica subsp. Brassica oleracea and one Bremia lactucae (lettuce) isolate were included. All downy mildews sporulated on A. thaliana, suggesting A. candida suppresses broad-spectrum downy mildew resistance. The white rust resistance gene, RAC5, is being investigated. The resistance phenotype associated with RAC5 seems not to involve a hypersensitive response. RAC5 has been mapped telomeric of nga106 on chromosome 5, in a region lacking NB-LRR genes, the most common structural class of resistance genes known in A. thaliana.

New resistances offer opportunity for effective management of the downy mildew (Hyaloperonospora parasitica) threat to canola

Downy mildew (Hyaloperonospora parasitica) is a problem for canola production worldwide, including in Australia where it has remained a persistent threat since 1998. Testing of 131 Brassicaceae varieties, including 109 Australian canola varieties (Brassica napus and B. juncea) and 22 diverse Brassicaceae (including B. napus, B. carinata, B. juncea, B. nigra, B. rapa, Crambe abyssinica and Raphanus sativus) highlighted excellent resistance to downy mildew. Using a mixture of 10 H. parasitica isolates, R. sativus Colonel and Boss showed highest resistance to H. parasitica, with per cent disease index (%DI) values of 3.7% and 10.2%, respectively. These were followed by (%DI values): B. carinata ATC 94011 (11.1%), B. napus CB™ Tanami (14.1%) and Komet-741 A (14.3%), B. juncea 397.23.2.3.3 (14.8%), B. napus ATR-Banjo (16.9%), Hyola 575 CL (16.9%), Komet-744 A (18.1%), Cresor-770 B (18.5%), Wamus (18.5%), Surpass 400 (19.2%), Hyola 432 (19.4%) and Hyola 76 (19.4%), and C. abyssinica (19.9%). These varieties were also considered highly resistant. Another five B. juncea genotypes and B. nigra P.23845 were considered highly resistant with %DI of 22.2%. Those considered resistant (but not highly resistant) included hybrid B. napus Hyola 444 TT, Hyola 500 RR, Hyola 504 RR, Pioneer 46Y78, Pioneer 45Y77 and Hyola 650 TT, and the non-hybrid variety ATR-Eyre, all with %DI values 23.1–28.2%. By contrast, B. napus Thunder TT, Hyola 450 TT and ATR-Grace were highly susceptible with %DI values of 90.3%, 88.2% and 81.7%, respectively. Cluster analysis revealed six distinct clusters (highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, very susceptible) for the tested Brassicaceae genotypes that, on average, showed similar responses within each cluster against H. parasitica based on their %DI values. From 2000 onwards (with the exception of Surpass 400), 10 B. napus varieties and one B. juncea released were classified as highly resistant; however, there was no overall correlation between year of variety release and level of resistance expressed against H. parasitica. This is the first study to demonstrate the existence of very high levels of pathotype-independent resistance in Australian canola varieties to H. parasitica. The most resistant varieties identified can be used in canola breeding programs and also directly deployed into regions where downy mildew is prevalent, providing the canola industry with an immediate and effective option for management of this important disease.

Use of rooted leaves for screening of Brassica germplasm response to clubroot (Plasmodiophora brassicae) and downy mildew (Hyaloperonospora parasitica)

Rooted leaves and cotyledons of various cruciferous crops were used for the screening of Brassica germplasm response to two obligatory pathogens: clubroot (Plasmodiophora brassicae) and downy mildew (Hyalope-ronospora parasitica). The development of roots was induced after 20-hour dipping of leaf pedicels in the mixture of growth regulators: indolyl-butyric acid (10 mg/l) and nicotinic acid (5 mg/l). The detached rooted leaves and cotyledons were maintained in 250ml plastic containers with perlite under fluorescent tubes in a growth chamber. With additional foliar fertilizing they remain vital for four months, producing clubroot galls on roots when dip-inoculated with Plasmodiophora spores and sporulating mycelia of downy mildew on leaves after drop inoculation with Hyaloperonospora parasitica. The possibilities of enhancing the sensitivity of this alternative assay in combination with immunochemical methods are discussed.