Many plant-associated microbes are pathogens that impair plant growth and reproduction. Plants respond to infection using a two-branched innate immune system. The first branch recognizes and responds to molecules common to many classes of microbes, including non-pathogens. The second responds to pathogen virulence factors, either directly or through their effects on host targets. These plant immune systems, and the pathogen molecules to which they respond, provide extraordinary insights into molecular recognition, cell biology and evolution across biological kingdoms. A detailed understanding of plant immune function will underpin crop improvement for food, fibre and biofuels production.

http://www.segenetica.es/cng2/cng2008/biblio/Ref-Marc-Valls---Jones-and-Dangl-Nat06.pdf

The plant immune system

Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

▪ Abstract Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS) Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp harbor large arsenals of effectors The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others Investigations of the functions of effector

/pdf/type-iii-secretion-system-effector-proteins-double-agents-in-tesdvhwezb.pdf

TYPE III SECRETION SYSTEM EFFECTOR PROTEINS: Double Agents in Bacterial Disease and Plant Defense

Animals and plants carry recognition systems to sense bacterial flagellin. Flagellin perception in Arabidopsis involves FLS2, a Leu-rich-repeat receptor kinase. We surveyed the early transcriptional response of Arabidopsis cell cultures and seedlings within 60 min of treatment with flg22, a peptide corresponding to the most conserved domain of flagellin. Using Affymetrix microarrays, approximately 3.0% of 8,200 genes displayed transcript level changes in flg22 elicited suspension cultures and seedlings. FLARE (Flagellin Rapidly Elicited) genes mostly encode signaling components, such as transcription factors, protein kinases/phosphatases, and proteins that regulate protein turnover. Approximately 80% of flg22-induced genes were also up-regulated in Arabidopsis seedlings treated with cycloheximide. This suggests that many FLARE genes are negatively regulated by rapidly turned-over repressor proteins. Twenty-one tobacco Avr9/Cf-9 rapidly elicited (ACRE) cDNA full-length sequences were used to search for their Arabidopsis orthologs (AtACRE). We identified either single or multiple putative orthologs for 17 ACRE genes. For 13 of these ACRE genes, at least one Arabidopsis ortholog was induced in flg22-elicited Arabidopsis suspension cells and seedlings. This result revealed a substantial overlap between the Arabidopsis flg22 response and the tobacco Avr9 race-specific defense response. We also compared FLARE gene sets and genes induced in basal or gene-for-gene interactions upon different Pseudomonas syringae treatments, and infer that Pseudomonas syringae pv tomato represses the flagellin-initiated defense response.

The Transcriptional Innate Immune Response to flg22. Interplay and Overlap with Avr Gene-Dependent Defense Responses and Bacterial Pathogenesis

Pseudomonas syringae is a Gram-negative, rod-shaped bacterium with polar flagella (Figure 1; Agrios, 1997). Strains of P. syringae collectively infect a wide variety of plants. Different strains of P. syringae, however, are known for their diverse and host-specific interactions with plants (Hirano and Upper, 2000). A specific strain may be assigned to one of at least 40 pathovars based on its host range among different plant species (Gardan et al., 1999) and then further assigned to a race based on differential interactions among cultivars of the host plant. Understanding the molecular basis of this high level of host specificity has been a driving force in using P. syringae as a model for the study of host-pathogen interactions. In crop fields, infected seeds are often an important source of primary inoculum in P. syringae diseases, and epiphytic bacterial growth on leaf surfaces often precedes disease development (Hirano and Upper, 2000). P. syringae enters the host tissues (usually leaves) through wounds or natural openings such as stomata, and in a susceptible plant it multiplies to high population levels in intercellular spaces. Infected leaves show water-soaked patches, which eventually become necrotic. Depending on P. syringae strains, necrotic lesions may be surrounded by diffuse chlorosis. Some strains of P. syringae also cause cankers and galls (Agrios, 1997). In resistant plants, on the other hand, P. syringae triggers the hypersensitive response (HR), a rapid, defense-associated death of plant cells in contact with the pathogen (Klement, 1963; Klement et al., 1964; Bent, 1996; Greenberg, 1996; Dangl et al., 1996; Hammond-Kosack and Jones, 1997). In this situation, P. syringae fails to multiply to high population levels and causes no disease symptoms.




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Figure 1.


A transmission electron microscope image of Pseudomonas syringae pv. tomato DC3000. Note that DC3000 produces polar flagella (15 nm in diameter) and a few Hrp pili (8 nm in diameter). The flagella and Hrp pili are indicated with arrows. Flagella enable bacteria to swim toward or away from specific chemical stimuli. Hrp pili are involved in type III secretion of avirulence and virulence proteins.

https://bioone.org/journals/The-Arabidopsis-Book/volume-2002/issue-1/tab.0039/The-Arabidopsis-Thaliana-Pseudomonas-Syringae-Interaction/10.1199/tab.0039.pdf

The Arabidopsis Thaliana-Pseudomonas Syringae Interaction

We have developed a model system to examine suppression of defense responses in bean by the compatible bacterium Pseudomonas syringae pv phaseolicola. Previously, we have shown that there is a general mechanism for the induction of the bean defense genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and chitinase (CHT) by incompatible, compatible, and nonpathogenic bacteria. Here, we show that bean plants infiltrated with isolates of P. s. phaseolicola failed to produce transcripts for PAL, CHS, or CHI up to 120 hr after infiltration and CHT transcript accumulation was significantly delayed when compared to the incompatible P. syringae strains. Infiltration of bean plants with 108 cells per mL of P. s. phaseolicola NPS3121 8 hr prior to infiltration with an equal concentration of incompatible P. s. pv tabaci Pt11528 significantly reduced the typical profile of defense transcript accumulation when compared to plants infiltrated with Pt11528 alone. A corresponding suppression of phytoalexin accumulation was also observed. NPS3121 also suppressed PAL, CHS, CHI, and CHT transcript accumulation and phytoalexin production induced by Escherichia coli DH5[alpha] or the elicitor glutathione. Heat-killed NPS3121 cells or cells treated with protein synthesis inhibitors lost the suppressor activity. Taken together, these experiments suggest that NPS3121 has an active mechanism to suppress the accumulation of defense transcripts and phytoalexin biosynthesis in bean.

Suppression of Bean Defense Responses by Pseudomonas syringae.

A number of inducible plant responses are believed to contribute to disease resistance. These responses include the hypersensitive reaction, phytoalexin synthesis, and the production of chitinase, glucanase, and hydroxyproline-rich glycoproteins. Because of the coordinate induction of these responses, it has been difficult to determine whether they are functional defense responses, and if they are, how they specifically contribute to disease resistance. Recent developments in molecular biology have provided experimental techniques that will reveal the specific contribution of each response to disease resistance. In this paper, we describe a strategy to determine if the hypersensitive reaction is a functional plant defense mechanism. Key words: chalcone isomerase, chalcone synthase, chitinase, hrp genes, hypersensitive reaction, nematode, phenylalanine ammonia-lyase, phytoalexin, Pseudomonas syringae, resistance.

Molecular analysis of plant defense responses to plant pathogens.

We describe a methodology utilizing high-throughput and high-content screening for novel adjuvant candidates that was used to screen a library of ∼2,500 small molecules via a 384-well quantitative combined cytokine and flow cytometry immunoassay in primary human peripheral blood mononuclear cells (PBMCs) from 4 healthy adult study participants. Hits were identified based on their induction of soluble cytokine (TNF, IFNγ and IL-10) secretion and PBMC maturation (CD 80/86, Ox40, and HLA-DR) in at least two of the four donors screened. From an initial set of 197 compounds identified using these biomarkers—an 8.6% hit rate—we downselected to five scaffolds that demonstrated robust efficacy and potency in vitro and evaluated the top hit, vinblastine sulfate, for adjuvanticity in vivo. Vinblastine sulfate significantly enhanced murine humoral responses to recombinant SARS-CoV-2 spike protein, including a four-fold enhancement of IgG titer production when compared to treatment with the spike antigen alone. Overall, we outline a methodology for discovering immunomodulators with adjuvant potential via high-throughput screening of PBMCs in vitro that yielded a lead compound with in vivo adjuvanticity. Motivation Vaccines are a key biomedical intervention to prevent the spread of infectious diseases, but their efficacy can be limited by insufficient immunogenicity and nonuniform reactogenic profiles. Adjuvants are molecules that potentiate vaccine responses by inducing innate immune activation. However, there are a limited number of adjuvants in approved vaccines, and current approaches for preclinical adjuvant discovery and development are inefficient. To enhance adjuvant identification, we developed a protocol based on in vitro screening of human primary leukocytes.

/pdf/adjuvant-discovery-via-a-high-throughput-screen-using-human-2aqy2dqf.pdf

Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells

The phytocannabinoid cannabidiol, an antiepileptic agent whose molecular targets are unclear, was found to potently enhance current carried by cloned human Kv7.2/7.3 channels and native M-current in mouse and rat neurons. 

Author response: Cannabidiol activates neuronal Kv7 channels

We have developed a model system to examine suppression of defense responses in bean by the compatible bacterium Pseudomonas syringae pv phaseolicola. Previously, we have shown that there is a general mechanism for the induction of the bean defense genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and chitinase (CHT) by incompatible, compatible, and nonpathogenic bacteria. Here, we show that bean plants infiltrated with isolates of R s. phaseolicola failed to produce transcripts for PAL, CHS, or CHI up to 120 hr after infiltration and CHT transcript accumulation was significantly delayed when compared to the incompatible R syringae strains. Infiltration of bean plants with 108 cells per mL of R s. phaseolicola NPS3121 8 hr prior to infiltration with an equal concentration of incompatible R s. pv tabaci Pt11528 significantly reduced the typical profile of defense transcript accumulation when compared to plants infiltrated with Pt11528 alone. A corresponding suppression of phytoalexin accumulation was also observed. NPS3121 also suppressed PAL, CHS, CHI, and CHT transcript accumulation and phytoalexin production induced by Escherichia col i DH5a or the elicitor glutathione. Heat-killed NPS3121 cells or cells treated with protein synthesis inhibitors lost the suppressor activity. Taken together, these experiments suggest that NPS3121 has an active mechanism to suppress the accumulation of defense transcripts and phytoalexin biosynthesis in bean.

Jennifer A. Smith

Papers

Suppression of Bean Defense Responses by Pseudomonas syringae.

Molecular analysis of plant defense responses to plant pathogens.

Adjuvant Discovery via a High Throughput Screen using Human Primary Mononuclear Cells

Author response: Cannabidiol activates neuronal Kv7 channels

Suppression of Bean Defense Pseudomonas syringae Responses bY