Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMVand mock-treated plants at 2, 6 and 24 h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

A decade ago, shoot proliferation symptoms (witches’ broom) in carrots were believed to be the cause of ‘Candidatus Phytoplasma’ and/or Spiroplasma infection, yet in recent years, this association appeared to have weakened and a closer association was found with the yet-unculturable, psyllid-transmitted Gram-negative bacterium, ‘Candidatus Liberibacter solanacearum’. In Israel, carrots are grown throughout the year, yet shoot proliferation symptoms tend to appear only in mature plants and mostly during late spring to early summer. We hypothesized that factors such as plant age, temperature and vector load, which vary along the year, have a critical effect on symptoms development and set to examine these factors under controlled conditions. Here we show that young carrot seedlings are as prone as older plants, to develop shoot proliferation symptoms, following ‘Ca. L. solanacearum’ inoculation. Surprisingly, we found that the local ‘Ca. L. solanacearum’ haplotype was extremely sensitive to constant temperature of 30˚C, which led to a significant reduction in bacterial growth and symptoms development, compared with 18˚C which was very conducive for symptoms development. We have also found that inoculations with 10 or 20 psyllids per plant results in faster symptoms development compared with inoculations with 2 psyllids per plant, however, the disease progress rate was insignificant among the different vector loads. These data provide important insight to the effects of plant age, temperature and vector load on ‘Ca. L. solanacearum’ and its associated symptoms and strengthen the notion that ‘Ca. L. solanacearum’ is the main responsible agent for carrot witches broom in Israel.

Gram negative bacteria form spherical blebs on their cell periphery, which later dissociate and released into the surrounding environment. Previous studies have shown that these nano scale structures, derived primarily from the bacterial outer membrane and are termed outer membrane vesicles (OMVs), induce typical immune outputs in both mammals and plants. On the other hand, these same structures have been shown to promote infection and disease. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated samples at 2, 6 and 24 h post challenge. We found that the most pronounced transcriptional shift occurred in the first two time points, as was reflected by both the number of differentially expressed genes (DEGs) and the average fold change. Gene ontology enrichment analysis revealed that OMVs induce a major transcriptional shift in Arabidopsis towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors and transcriptional factors. Comparing Arabidopsis response to OMVs and to single purified elicitors, revealed that while OMVs induce a similar suite of genes and pathways as single elicitors, some differential pathways activated by OMVs were detected including response to drug and apoptosis, which may indicate exposure to toxic compounds via OMV. To examine whether the observed transcriptional shift in Arabidopsis leads to an effective immune response, plants were pretreated with OMVs and then inoculated with a bacterial pathogen. OMV-mediated priming led to a significant reduction in bacterial titer in inoculated leaves two days following inoculation. Mutations in the elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) receptor, did not significantly affect OMV-priming. All together these results show that OMV induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change is reflected in the ability to better resist bacterial infection.