Rhizobacteria Bacillus subtilis restricts foliar pathogen entry through stomata

Plants exist in a complex multitrophic environment, where they interact with and compete for resources with other plants, microbes and animals. Plants have a complex array of defense mechanisms, such as the cell wall being covered with a waxy cuticle serving as a potent physical barrier. Although some pathogenic fungi infect plants by penetrating through the cell wall, many bacterial pathogens invade plants primarily through stomata on the leaf surface. Entry of the foliar pathogen, Pseudomonas syringae pathovar tomato DC3000 (hereafter PstDC3000), into the plant corpus occurs through stomatal openings, and consequently a key plant innate immune response is the transient closure of stomata, which delays disease progression. Here, we present evidence that the root colonization of the rhizobacteria Bacillus subtilis FB17 (hereafter FB17) restricts the stomata‐mediated pathogen entry of PstDC3000 in Arabidopsis thaliana. Root binding of FB17 invokes abscisic acid (ABA) and salicylic acid (SA) signaling pathways to close light‐adapted stomata. These results emphasize the importance of rhizospheric processes and environmental conditions as an integral part of the plant innate immune system against foliar bacterial infections.     

Role of plant stomata in bacterial invasion

Stomata are microscopic pores in the epidermis of the aerial parts of terrestrial plants. These pores are essential for photosynthesis, as they allow CO(2) to diffuse into the plant. The size of the stomatal pore changes in response to environmental conditions, such as light intensity, air humidity and CO(2) concentrations, as part of the plant's adaptation to maximize photosynthetic efficiency and, at the same time, to minimize water loss. Historically, stomata have been considered as passive portal of entry for plant pathogenic bacteria. However, recent studies suggest that stomata can play an active role in restricting bacterial invasion as part of the plant innate immune system. Some plant pathogens have evolved specific virulence factors to overcome stomata-based defence. Interestingly, many bacterial disease outbreaks require high humidity, rain, or frost damage, which could promote stomatal opening and/or bypass stomatal defence by creating wounds as alternative entry sites. Further studies on microbial and environmental regulation of stomata-based defence should fill gaps in our understanding of bacterial pathogenesis, disease epidemiology and phyllosphere microbiology.     

气孔免疫的研究进展及展望

气孔长期以来被认为是植物病原菌入侵植物体内的被动通道,而最新的研究则表明气孔作为植物先天免疫的重要环节,在限制细菌入侵方面起到主动作用。这一发现也带动了植物免疫学,即植物气孔开合调控和植物免疫学交叉学科的快速发展。基于此,本文对气孔免疫的机制研究展开综述并对其对植物水分利用效率的影响进行展望。     

Bacterial infection systemically suppresses stomatal density

Many plant pathogens gain entry to their host via stomata. On sensing attack, plants close these pores to restrict pathogen entry. Here, we show that plants exhibit a second longer term stomatal response to pathogens. Following infection, the subsequent development of leaves is altered via a systemic signal. This reduces the density of stomata formed, thus providing fewer entry points for pathogens on new leaves. Arabidopsis thaliana leaves produced after infection by a bacterial pathogen that infects through the stomata (Pseudomonas syringae) developed larger epidermal pavement cells and stomata and consequently had up to 20% reductions in stomatal density. The bacterial peptide flg22 or the phytohormone salicylic acid induced similar systemic reductions in stomatal density suggesting that they might mediate this effect. In addition, flagellin receptors, salicylic acid accumulation, and the lipid transfer protein AZI1 were all required for this developmental response. Furthermore, manipulation of stomatal density affected the level of bacterial colonization, and plants with reduced stomatal density showed slower disease progression. We propose that following infection, development of new leaves is altered by a signalling pathway with some commonalities to systemic acquired resistance. This acts to reduce the potential for future infection by providing fewer stomatal openings.     

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.     

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H⁺-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H⁺-ATPase activity. PM H⁺-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H⁺-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H⁺-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.     

RIN4 Functions with Plasma Membrane H+-ATPases to Regulate Stomatal Apertures during Pathogen Attack

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H⁺-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H⁺-ATPase activity. PM H⁺-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H⁺-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H⁺-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation

Stomata in the epidermis of terrestrial plants are important for CO2 absorption and transpirational water loss, and are also potential points of entry for pathogens. Stomatal opening and closure are controlled by distinct mechanisms. Arabidopsis stomata have been shown to close in response to bacteria and pathogen-associated molecular patterns (PAMPs) as part of PAMP-triggered immunity (PTI). Here we show that flg22, a PAMP derived from bacterial flagellin, also inhibits light-induced stomatal opening. Consistent with our observations on stomatal opening, flg22 inhibits the inward K+ channels (K+ (in) currents) of guard cells that mediate K+ uptake during stomatal opening. Similar to previously documented K+ current changes triggered by exogenous elevation of H(2)O(2) and nitric oxide (NO), with prolonged duration of flg22 exposure the outward K+ channels (K+ (out) currents) of guard cells are also inhibited. In null mutants of the flg22 receptor, FLS2, flg22 regulation of stomatal opening, K+ (in) currents, and K+ (out) currents is eliminated. flg22 also fails to elicit these responses in null mutants of the sole canonical G-protein alpha subunit, GPA1. The bacterial toxin, coronatine, produced by several pathogenic strains of Pseudomonas syringae, reverses the inhibitory effects of flg22 on both K+ (in) currents and stomatal opening, indicating interplay between plant and pathogen in the regulation of plant ion channels. Thus, the PAMP-triggered stomatal response involves K+ channel regulation, and this regulation is dependent on signaling via cognate PAMP receptors and a heterotrimeric G-protein. These new findings provide insights into the largely elusive signaling process underlying PTI-associated guard cell responses.     

The Xanthomonas type Ⅲ effector Xop AP prevents stomatal closure by interfering with vacuolar acidification

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid(ABA) or salicylic acid(SA) and after recognition of pathogenassociated molecular patterns(PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type Ⅲ effectors(T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas...     

The Arabidopsis Lectin Receptor Kinase LecRK-V.5 Represses Stomatal Immunity Induced by Pseudomonas syringae pv. tomato DC3000

Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO₂ uptake and photosynthesis.     

Stomata Prioritize Their Responses to Multiple Biotic and Abiotic Signal Inputs

Stomata are microscopic pores in leaf epidermis that regulate gas exchange between plants and the environment. Being natural openings on the leaf surface, stomata also serve as ports for the invasion of foliar pathogenic bacteria. Each stomatal pore is enclosed by a pair of guard cells that are able to sense a wide spectrum of biotic and abiotic stresses and respond by precisely adjusting the pore width. However, it is not clear whether stomatal responses to simultaneously imposed biotic and abiotic signals are mutually dependent on each other. Here we show that a genetically engineered Escherichia coli strain DH5α could trigger stomatal closure in Vicia faba, an innate immune response that might depend on NADPH oxidase-mediated ROS burst. DH5α-induced stomatal closure could be abolished or disguised under certain environmental conditions like low [CO₂], darkness, and drought, etc. Foliar spraying of high concentrations of ABA could reduce stomatal aperture in high humidity-treated faba bean plants. Consistently, the aggressive multiplication of DH5α bacteria in Vicia faba leaves under high humidity could be alleviated by exogenous application of ABA. Our data suggest that a successful colonization of bacteria on the leaf surface is correlated with stomatal aperture regulation by a specific set of environmental factors.     

盾叶秋海棠叶表皮气孔簇的发育及分布格局

气孔是植物控制气体交换和调节水分散失的门户。大部分高等植物气孔的分布格局是相邻气孔之间被一至多个表皮细胞所间隔。而在有限分布的几个科属的植物种中发现气孔成簇分布的现象 ,即由 2至多个紧密相邻的气孔器组成相对独立的单元 ,称为气孔簇 (stomatalcluster)。以中国原产的盾叶秋海棠 (BegoniapeltatifoliaLi)为研究对象 ,探讨了叶表皮气孔簇的发育机制及其分布格局。结果表明 :气孔发育初期 ,气孔拟分生组织的成簇 (相邻紧密 )排列可能是气孔簇形成的主要机制 ;气孔副卫细胞恢复分裂形成的卫星拟分生组织也对气孔簇的形成起一定的作用。把气孔簇和单个气孔视为一个气孔单元发现 ,盾叶秋海棠气孔单元密度 (单位面积中气孔单元数 )和气孔单元大小 (气孔单元所包含气孔数 )在叶片上呈有规律的分布 :前者由叶片中部向叶尖、叶缘逐圈增多 ,而后者逐圈减少。对这种分布格局的成因进行了讨论     

Immunity of a leguminous plant infected by nodular bacteria <Emphasis Type="Italic">Rhizobium</Emphasis> spp. F.: Review

Recent studies of the immune system of leguminous plants infected with nodular bacteria (rhizobia) are summarized. The possibility of blocking the invasion of rhizobia into plant organs not affected by the primary infection is discussed. The concept of local and systemic resistance of the leguminous plant to rhizobial infection is introduced. The Nod factors of rhizobia are considered, as well as the plant receptors that interact with these factors upon the formation of symbiosis of the plant and bacteria. The role of bacterial surface exopolysaccharides in the suppression of the protective system of the plants is discussed. The innate immunity of leguminous plant cells is assumed to affect the formation and functioning of the symbiosis of the plant and the bacteria.     

Autophagy and plant innate immunity

Plant innate immunity is often associated with specialized programmed cell death at or near the site of pathogen infection. Despite the isolation of several lesion mimic mutants, the molecular mechanisms that regulate cell death during an immune response remain obscure. Recently, autophagy, an evolutionarily conserved process of bulk protein and organelle turnover, was shown to play an important role in limiting cell death initiated during plant innate immune responses. Consistent with its role in plants, several studies in animals also demonstrate that the autophagic machinery is involved in innate as well as adaptive immunities. Here, we review the role of autophagy in plant innate immunity. Because autophagy is observed in healthy and dying plant cells, we will also examine whether autophagy plays a protective or a destructive role during an immune response.     

Human serum amyloid A protein inhibits hepatitis C virus entry into cells

Serum amyloid A (SAA) is an acute-phase protein induced by a variety of inflammatory stimuli, including bacterial and viral infections. SAA was recently found to function as an opsonin for gram-negative bacteria. We report here that SAA inhibited hepatitis C virus (HCV) infection in cultured cells. SAA reduced HCV infectivity in a dose-dependent manner when added during HCV infection but not after virus entry. SAA bound HCV virions and specifically blocked HCV entry but did not affect virus attachment. These findings suggest that SAA functions as part of the host innate immune defense mechanisms against HCV infection in humans.     

Stomatal responses to changes in temperature at increasing water stress

Summary The response of stomata to a gradual increase in temperature at increasing plant water stress was studied in a hot desert habitat (Negev, Israel) in the field, but under controlled temperature and humidity conditions. Four native species (Zygophyllum dumosum, Artemisia herba-alba, Hammada scoparia, Reaumuria negevensis) and one cultivated plant (Prunus armeniaca) were used in these studies. The stomatal response to temperature was compared with the response in well-irrigated plants of the same species.At low water stress, the diffusion resistance for water vapour decreased in response to a gradual increase in temperature. Transpiration increased accordingly. This response was reversible. All species responded in the same way. The opening of stomata with increasing temperature was apparently independent of the stomatal response regulated by atmospheric humidity. At high plant water stress, the stomatal response was reversed, i.e., the stomata closed when temperature was gradually increased. This stomatal closure was also independent of the closure regulated by atmospheric humidity. The plant water potential at which the stomatal response to temperature was reversed, differed among the species investigated.