The hemibiotrophic fungal pathogen Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus (canola, oilseed rape) and causes significant loss of yield worldwide. While genetic resistance has been used to mitigate the disease by means of traditional breeding strategies, there is little knowledge about the genes that contribute to blackleg resistance. RNA sequencing and a streamlined bioinformatics pipeline identified unique genes and plant defense pathways specific to plant resistance in the B. napus-L. maculans LepR1-AvrLepR1 interaction over time. We complemented our temporal analyses by monitoring gene activity directly at the infection site using laser microdissection coupled to quantitative PCR. Finally, we characterized genes involved in plant resistance to blackleg in the Arabidopsis-L. maculans model pathosystem. Data reveal an accelerated activation of the plant transcriptome in resistant host cotyledons associated with transcripts coding for extracellular receptors and phytohormone signaling molecules. Functional characterization provides direct support for transcriptome data and positively identifies resistance regulators in the Brassicaceae. Spatial gradients of gene activity were identified in response to L. maculans proximal to the site of infection. This dataset provides unprecedented spatial and temporal resolution of the genes required for blackleg resistance and serves as a valuable resource for those interested in host-pathogen interactions.

Our study investigated disease resistance in the Brassica napus–Leptosphaeria maculans pathosystem using a combination of laser microdissection, dual RNA sequencing, and physiological validations of large-scale gene sets. The use of laser microdissection improved pathogen detection and identified putative L. maculans effectors and lytic enzymes operative during host colonization. Within 24 h of inoculation, we detected large shifts in gene activity in resistant cotyledons associated with jasmonic acid and calcium signaling pathways that accelerated the plant defense response. Sequencing data were validated through the direct quantification of endogenous jasmonic acid levels. Additionally, resistance against L. maculans was abolished when the calcium chelator EGTA was applied to the inoculation site, providing physiological evidence of the role of calcium in B. napus immunity against L. maculans. We integrated gene expression data with all available information on cis-regulatory elements and transcription factor binding affinities to better understand the gene regulatory networks underpinning plant resistance to hemibiotrophic pathogens. These in silico analyses point to early cellular reprogramming during host immunity that are coordinated by CAMTA, BZIP, and bHLH transcription factors. Together, we provide compelling genetic and physiological evidence into the programming of plant resistance against fungal pathogens.

The microbial composition of the rhizosphere soil could be an important determinant of crop yield, pathogen resistance, and other beneficial attributes in plants. However, little is known about the impact of cropping sequences on microbial community dynamics, especially in economically important species like soybean. Using 2-year crop sequences of corn-soybean, canola-soybean, and soybean-soybean, we investigated how crops from the previous growing season influenced the structure of the microbiome in both the bulk soil and soybean rhizosphere. A combination of marker-based Illumina sequencing and bioinformatics analyses was used to show that bacterial species richness and evenness in the soybean rhizosphere soil were similar following canola and soybean compared to a previous corn sequence. However, fungal species richness and evenness remained unaffected by crop sequence. In addition, bacterial and fungal species diversity in both the bulk and soybean rhizosphere soil were not influenced by crop sequence. Lastly, the corn-soybean sequence significantly differed in the relative abundance of certain bacterial and fungal classes in both the soybean rhizosphere and bulk soil. While canola-soybean and a continuous soybean sequence did not, suggesting that a preceding corn sequence may reduce the occurrence of overall bacterial and fungal community members. For the present study, crop sequence impacts bacterial diversity and richness in both the bulk soil and soybean rhizosphere soil whereas fungal diversity and richness are resilient to crop sequence practices. Together, these findings could help drive decision making for annual crop and soil management practices.

The causative agent of white mold, Sclerotinia sclerotiorum, is capable of infecting over 600 plant species and is responsible for significant crop losses across the globe. Control is currently dependent on broad-spectrum chemical agents that can negatively impact the agroecological environment, presenting a need to develop alternative control measures. In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing hairpin (hp)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 compared to wild-type Col-0. To better understand how HIGS influences host–pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 directly at the site of S. sclerotiorum infection. RNA sequencing data reveals enrichment of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) pathway, as well as transcription factors predicted to regulate plant immunity. Using RT-qPCR, we identified predicted interacting partners of ABHYDROLASE-3 in the polyamine synthesis pathway of S. sclerotiorum that demonstrate co-reduction with ABHYDROLASE-3 transcript levels during infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provide insight into the role of ABHYDROLASE-3 in the A. thaliana–S. sclerotiorum pathosystem.

The causative agent of white mold, Sclerotinia sclerotiorum, is capable of infecting over 600 plant species and is responsible for significant crop losses across the globe. Control is currently dependent on broad-spectrum chemical agents that can negatively impact the agroecological environment, presenting a need to develop alternative control measures. In this study, we developed transgenic Arabidopsis thaliana (AT1703) expressing double-stranded (ds)RNA to silence S. sclerotiorum ABHYDROLASE-3 and slow infection through host induced gene silencing (HIGS). Leaf infection assays show reduced S. sclerotiorum lesion size, fungal load, and ABHYDROLASE-3 transcript abundance in AT1703 compared to wild-type Col-0. To better understand how HIGS influences host-pathogen interactions, we performed global RNA sequencing on AT1703 and wild-type Col-0 directly at the site of S. sclerotiorum infection. RNA sequencing data reveals enrichment of the salicylic acid (SA)-mediated systemic acquired resistance (SAR) pathway, as well as transcription factors predicted to regulate plant immunity. Using qPCR, we identified predicted interacting partners of ABHYDROLASE-3 in the polyamine synthesis pathway of S. sclerotiorum that demonstrate co-reduction with ABHYDROLASE-3 transcript levels during infection. Together, these results demonstrate the utility of HIGS technology in slowing S. sclerotiorum infection and provide insight into the role of ABHYDROLASE-3 in the A. thaliana – S. sclerotiorum pathosystem.