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Abstract Over 110 million tons of nitrogen fertilizer every year is used for crop production. Scientists have dreamed of enabling rhizobial nitrogen fixation in non-leguminous crops to mitigate the increasing demand for nitrogen fertilizer. However, despite decades of research, rhizobial nitrogen fixation in non-host plants has not been demonstrated. Here, we reported that an N-fixing rhizobium and a clubroot pathogen Plasmodiophora brassicae exhibited a synergistic effect on fixing nitrogen in cruciferous plants. Rhizobia were found to invade P. brassicae-infected rapeseed (Brassica napus) roots in the field. The colonization of rhizobium on rapeseed roots was confirmed by co-inoculating Mesorhizobium huakuii with P. brassicae under controlled laboratory conditions. M. huakuii infection could alleviate clubroot symptoms and promote the growth of diseased rapeseeds. M. huakuii could fix nitrogen in P. brassicae-infected plants based on the results of 15N isotope dilution tests. The expression of homologs of legume genes required for symbiosis and early-nodulin genes was significantly upregulated in Arabidopsis during early infection by P. brassicae. More importantly, M. huakuii could even fix nitrogen in P. brassicae-resistant rapeseed cultivar and promote plant growth when co-inoculated with P. brassicae. Our findings provide a new avenue to understand the interaction of rhizobia with non-host plants, stimulate the exploration of fixing nitrogen in non-leguminous plants by nitrogen-fixing rhizobia, and develop a strategy for both disease control and nitrogen fixation on non-host crops.

Abstract Background Enhancers are cis-regulatory elements present in eukaryote genomes, which constitute indispensable determinants of gene regulation by governing the spatiotemporal and quantitative expression dynamics of target genes, and are involved in multiple life processes, for instance during development and disease states. The importance of enhancer activity has additionally been highlighted for immune responses in animals and plants; however, the dynamics of enhancer activities and molecular functions in plant innate immunity are largely unknown. Here, we investigated the involvement of distal enhancers in early innate immunity in Arabidopsis thaliana. Results A group of putative distal enhancers producing low-abundance transcripts either unidirectionally or bidirectionally are identified. We show that enhancer transcripts are dynamically modulated in plant immunity triggered by microbe-associated molecular patterns and are strongly correlated with open chromatin, low levels of methylated DNA, and increases in RNA polymerase II targeting and acetylated histone marks. Dynamic enhancer transcription is correlated with target early immune gene expression patterns. Cis motifs that are bound by immune-related transcription factors, such as WRKYs and SARD1, are highly enriched within upregulated enhancers. Moreover, a subset of core pattern-induced enhancers are upregulated by multiple patterns from diverse pathogens. The expression dynamics of putative immunity-related enhancers and the importance of WRKY binding motifs for enhancer function were also validated. Conclusions Our study demonstrates the general occurrence of enhancer transcription in plants and provides novel information on the distal regulatory landscape during early plant innate immunity, providing new insights into immune gene regulation and ultimately improving the mechanistic understanding of the plant immune system.

Abstract Sclerotial development is a vital stage in the life cycles of many fungal plant pathogens. In this study, the protein Ss-CAD, which contains three conserved domains of cinnamyl alcohol dehydrogenase (CAD), was found to be required for sclerotial development in Sclerotinia sclerotiorum. Ss-CAD was significantly upregulated during early stage of sclerotial development. In Ss-CAD-silenced strains, sclerotial development was abnormal. In these silenced strains, formation of sclerotia was delayed or sclerotia yield was reduced, whereas hyphal growth and virulence were normal. Nox1, Nox2, and NoxR, which encode reactive oxygen species (ROS)-generating NADPH oxidases, were downregulated in Ss-CAD-silenced strains. NoxR-silenced strains displayed similar defects during sclerotial formation as Ss-CAD-silenced strains. Treatment of Ss-CAD-silenced strains with exogenous oxidants or NADPH restored normal sclerotial development. Sclerogenesis in Ss-CAD-silenced strains could also be recovered through Nox1 overexpression. The results suggest that Ss-CAD is linked to the NADPH oxidase pathways to affect sclerotial development in S. sclerotiorum.

Abstract Plasmodiophora brassicae is an important biotrophic eukaryotic plant pathogen and a member of the rhizarian protists. This biotrophic pathogen causes clubroot in cruciferous plants via novel intracellular mechanisms that are markedly different from those of other biotrophic organisms. To date, genomes from six single spore isolates of P. brassicae have been sequenced. An accurate description of the evolutionary status of this biotrophic protist, however, remains lacking. Here, we determined the draft genome of the P. brassicae ZJ-1 strain. A total of 10,951 protein-coding genes were identified from a 24.1 Mb genome sequence. We applied a comparative genomics approach to prove the Rhizaria supergroup is an independent branch in the eukaryotic evolutionary tree. We also found that the GPCR signaling pathway, the versatile signal transduction to multiple intracellular signaling cascades in response to extracellular signals in eukaryotes, is significantly enriched in P. brassicae-expanded and P. brassicae-specific gene sets. Additionally, treatment with a GPCR inhibitor relieved the symptoms of clubroot and significantly suppressed the development of plasmodia. Our findings suggest that GPCR signal transduction pathways play important roles in the growth, development, and pathogenicity of P. brassicae.

Abstract Background The eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear. Results Here, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia. Conclusions Proto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

Following the publication of this article [1], the authors noted the following errors.