Browsing by Subject "plant immunity"

Cells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.

Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation.

Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.

The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.

The activation and maintenance of plant immune responses require a significant amount of energy because they are accompanied by massive transcriptional reprogramming. Spurious activation of plant defense machinery can lead to autoimmune diseases and growth inhibition. So it is important for plants to tightly regulate the immune system to ensure the balance between growth and defense. However, neither the molecular mechanisms nor the design principles of how plants reach this balance are understood.

In this dissertation work, I showed how transcriptional and translational control of plant immune system can help avoid the constant immune surveillance and elicit a proper level of defense responses when necessary. These fine tunings of the immune system ensure the balance between growth and defense.

My research on the transcriptional regulation of plant defense responses led to the surprising discovery that even without pathogen, plant can 'anticipate' potential infection according to a circadian schedule under conditions that favor the initiation of infection. Functional analysis of 22 novel immune components unveiled their transient expression at dawn, when the infection is most likely to happen. This pulse expression pattern was shown to be regulated by the central circadian oscillator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) since these 22 genes are no longer induced in the cca1 mutant. Moreover, the temporal control of the transcription level of these 22 immune genes by CCA1 also fine tunes their expression pattern according to the perceptions of different pathogenic signals. At the basal defense level, the expression of these genes can be transiently induced upon perceptions of critical infection stages of the pathogen. When an elevated level of defense response is needed, the high expression levels of these genes are maintained to confer a stronger immunity against pathogen. Since this stronger form of defense may also cause the suicidal death of the plant cells, the interplay between the circadian clock and defense allows a better decision on the proper level of the immunity to minimize the sacrificial death. The circadian clock is also known to regulate the growth-related cellular functions extensively. So the circadian clock can help to balance the energy used in growth and defense through transcriptional regulation on both sides.

Besides the integrated control by the circadian clock, the translational control on a key transcription factor involved in the growth-to-defense transition can also maintain the balance between growth and defense.TBF1 is a major transcription factor that can initiate the growth-to-defense transition through transcriptional repression of growth-associated cellular functions and induction of defense-related machinery. Bioinformatics studies identified 2 upstream open reading frames (uORFs) encoding multiple phenylalanine at 5' of the translation initiation codon of TBF1. Under normal conditions, these 2 uORFs can repress the translation of TBF1 to prevent accidental activation. However, pathogen infection may cause rapid and transient depletion of phenylalanine, a well-known precursor for cell wall components and the SAR signal SA. This depletion signal can be reflected by the increase of uncharged tRNAPhe, which subsequently leads to the phosphorylation of eIF2á and the release of uORFs' repression on TBF1. These findings provided the molecular details of how uORF-based translational control can couple transcriptional reprogramming with metabolic status to coordinately trigger the growth-to-defense transition.

In summary, my dissertation work has identified previously unrecognized regulatory mechanisms by which plant immune responses are balanced with growth. These new findings will further investigations into these novel interfaces between plants and pathogens. Future studies will definitely further improve our understandings of the plant-microbe interactions.

Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.

I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.

Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.

In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.