Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria.

Plant immunity to avirulent bacterial pathogens is associated with subcellular membrane dynamics including fusion between the vacuolar and plasma membranes, resulting in hypersensitive cell death. Here, we report that ADAPTOR PROTEIN COMPLEX-4 (AP-4) subunits are involved in plant immunity associated with hypersensitive cell death. We isolated a mutant with a defect in resistance to an avirulent strain of Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpm1 from a vacuolar protein sorting mutant library of Arabidopsis (Arabidopsis thaliana). The mutant was identical to gfs4-1, which has a mutation in the gene encoding the AP-4 subunit AP4B. Thus, we focused on AP4B and another subunit, AP4E. All of the mutants (ap4b-3, ap4b-4, ap4e-1, and ap4e-2) were defective in hypersensitive cell death and resistance to Pto DC3000 with the type III effector AvrRpm1 or AvrRpt2, both of which are recognized on the plasma membrane, while they showed slightly enhanced susceptibility to the type-III-secretion-deficient P. syringae strain hrcC On the other hand, both ap4b-3 and ap4b-4 showed no defect in resistance to Pto DC3000 with the type III effector AvrRps4, which is recognized in the cytosol and does not induce hypersensitive cell death. Upon infection with Pto DC3000 avrRpt2, the ap4b-3 and ap4b-4 leaf cells did not show fusion between vacuolar and plasma membranes, whereas the wild-type leaf cells did. These results suggest that AP-4 contributes to cell death-associated immunity, possibly via membrane fusion, after type III effector-recognition on the plasma membrane.

A novel membrane fusion-mediated plant immunity against bacterial pathogens.

Plants have developed their own defense strategies because they have no immune cells. A common plant defense strategy involves programmed cell death (PCD) at the infection site, but how the PCD-associated cell-autonomous immunity is executed in plants is not fully understood. Here we provide a novel mechanism underlying cell-autonomous immunity, which involves the fusion of membranes of a large central vacuole with the plasma membrane, resulting in the discharge of vacuolar antibacterial proteins to the outside of the cells, where bacteria proliferate. The extracellular fluid that was discharged from the vacuoles of infected leaves had both antibacterial activity and cell death-inducing activity. We found that a defect in proteasome function abolished the membrane fusion associated with both disease resistance and PCD in response to avirulent bacterial strains but not to a virulent strain. Furthermore, RNAi plants with a defective proteasome subunit PBA1 have reduced DEVDase activity, which is an activity associated with caspase-3, one of the executors of animal apoptosis. The plant counterpart of caspase-3 has not yet been identified. Our results suggest that PBA1 acts as a plant caspase-3-like enzyme. Thus, this novel defense strategy through proteasome-regulating membrane fusion of the vacuolar and plasma membranes provides plants with a mechanism for attacking intercellular bacterial pathogens.

Constitutive and inducible ER bodies of Arabidopsis thaliana accumulate distinct beta-glucosidases.

The endoplasmic reticulum (ER) body is an ER-related organelle that accumulates high levels of PYK10, a beta-glucosidase with an ER retention signal. Constitutive ER bodies are present in the epidermal cells of cotyledons, hypocotyls and roots of young Arabidopsis seedlings, but absent in rosette leaves. When leaves are wounded, ER bodies are induced around the wounding site of the leaves (inducible ER bodies). To clarify the functional differences between these two ER bodies, we compared constitutive ER bodies with inducible ER bodies in wounded cotyledons of Arabidopsis seedlings. We found that the number of ER bodies increased both in cotyledons wounded directly (locally wounded cotyledons) and in unwounded cotyledons exposed to the systemic wound response (systemically wounded cotyledons). Quantitative reverse transcription-PCR and immunoblot analyses revealed that BGLU18, encoding another beta-glucosidase with an ER retention signal, was induced at the site of wounding, whereas PYK10 was not. Immunocytochemical analysis showed that BGLU18 protein was exclusively localized in ER bodies formed directly at the wounding site on cotyledons. ER bodies were not induced in locally and systemically wounded cotyledons of the bglu18 knock-out mutant. These results indicate that constitutive and inducible ER bodies accumulate different sets of beta -glucosidases and may have distinct functions in defense responses.

Hexose Oxidase-Mediated Hydrogen Peroxide as a Mechanism for the Antibacterial Activity in the Red Seaweed Ptilophora subcostata.

Marine algae have unique defense strategies against microbial infection. However, their mechanisms of immunity remain to be elucidated and little is known about the similarity of the immune systems of marine algae and terrestrial higher plants. Here, we suggest a possible mechanism underlying algal immunity, which involves hexose oxidase (HOX)-dependent production of hydrogen peroxide (H2O2). We examined crude extracts from five different red algal species for their ability to prevent bacterial growth. The extract from one of these algae, Ptilophora subcostata, was particularly active and prevented the growth of gram-positive and -negative bacteria, which was completely inhibited by treatment with catalase. The extract did not affect the growth of either a yeast or a filamentous fungus. We partially purified from P. subcostata an enzyme involved in its antibacterial activity, which shared 50% homology with the HOX of red seaweed Chondrus crispus. In-gel carbohydrate oxidase assays revealed that P. subcostata extract had the ability to produce H2O2 in a hexose-dependent manner and this activity was highest in the presence of galactose. In addition, Bacillus subtilis growth was strongly suppressed near P. subcostata algal fronds on GYP agar plates. These results suggest that HOX plays a role in P. subcostata resistance to bacterial attack by mediating H2O2 production in the marine environment.

A red-emitting ratiometric fluorescent probe based on a benzophosphole P-oxide scaffold for the detection of intracellular sodium ions.

We disclose the development of a ratiometric fluorescent probe based on a benzophosphole P-oxide and its application for the detection of intracellular Na(+) ions. Excitation by visible light induced red emission from this probe in water, which was subjected to a hypsochromic shift upon complexation with Na(+). Based on this change, a ratiometric analysis enabled us to visualise changes in the Na(+) concentration in living mammalian cells.

The ER body, a new organelle in Arabidopsis thaliana, requires NAI2 for its formation and accumulates specific beta-glucosidases.

Plants develop various ER-derived structures with specific functions. The ER body found in Arabidopsis thaliana is a spindle-shaped structure. ER bodies accumulate in epidermal cells in seedlings or are induced by wounding. The molecular mechanisms underlying the formation of the ER body remained obscure. We isolated an ER body-deficient mutant in Arabidopsis seedlings, which we termed nai2. The NAI2 gene encodes a member of a unique protein family. NAI2 localizes to the ER body and the downregulation of NAI2 elongates ER bodies and reduces their number. ER bodies specifically accumulate high levels of PYK10/BGLU23, which is a beta-glucosidase that bears an ER retention signal. Additionally, in the nai2 mutant, PYK10 protein is diffuse throughout the ER and the PYK10 protein level is reduced. These observations indicate that NAI2 is a key factor for the formation of ER bodies and for the accumulation of PYK10 in the ER bodies of Arabidopsis. We also found that BGLU18, which encodes another beta-glucosidase with an ER retention signal, is induced at the site of wounding. Immunocytochemical analysis revealed that the BGLU18 protein is exclusively localized in ER bodies formed directly at the wounding site of cotyledons. These results suggest that BGLU18 is a component of the ER body in wounded leaves of Arabidopsis.