An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2

Chloroplasts adapt to freezing and other abiotic stresses in part by modifying their membranes. One key-remodeling enzyme is SENSITIVE TO FREEZING2 (SFR2). SFR2 is unusual because it does not respond to initial cold stress or cold acclimation, instead it responds during freezing conditions in Arabidopsis. This response has been shown to be sensitive to cytosolic acidification. The unique lipid products of SFR2 have also been detected in response to non-freezing stresses, but what causes SFR2 to respond in these stresses is unknown. Here, we investigate protoplast isolation as a representative of wounding stress. We show that SFR2 oligogalactolipid products accumulate during protoplast isolation. Notably, we show that protoplast cytosol is acidified during isolation. Modification of the buffers reduces oligogalactolipid accumulation, while prolonged incubation in the isolated state increases it. We conclude that SFR2 activation during protoplast isolation correlates with cytosolic acidification, implying that all SFR2 activation may be dependent on cytosolic acidification. We also conclude that protoplasts can be more gently isolated, reducing their stress

The \u3ci\u3ePseudomonas syringae\u3c/i\u3e type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity

The \u3ci\u3eMagnaporthe oryzae\u3c/i\u3e nitrooxidative stress response suppresses rice innate immunity during blast disease

Understanding how microorganisms manipulate plant innate immunity and colonize host cells is a major goal of plant pathology. Here, we report that the fungal nitrooxidative stress response suppresses host defenses to facilitate the growth and development of the important rice pathogen Magnaporthe oryzae in leaf cells. Nitronate monooxygenases encoded by NMO genes catalyze the oxidative denitrification of nitroalkanes. We show that the M. oryzae NMO2 gene is required for mitigating damaging lipid nitration under nitrooxidative stress conditions and, consequently, for using nitrate and nitrite as nitrogen sources. On plants, the Δnmo2 mutant strain penetrated host cuticles like wild type, but invasive hyphal growth in rice cells was restricted and elicited plant immune responses that included the formation of cellular deposits and a host reactive oxygen species burst. Development of the M. oryzae effector-secreting biotrophic interfacial complex (BIC) was misregulated in the Δnmo2 mutant. Inhibiting or quenching host reactive oxygen species suppressed rice innate immune responses and allowed the Δnmo2 mutant to grow and develop normally in infected cells. NMO2 is thus essential for mitigating nitrooxidative cellular damage and, in rice cells, maintaining redox balance to avoid triggering plant defenses that impact M. oryzae growth and BIC development

Phylloquinone (vitamin K 1 ) biosynthesis in plants: two peroxisomal thioesterases of lactobacillales origin hydrolyze 1,4‐dihydroxy‐2‐naphthoyl‐coa

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/1/TPJ_4972_sm_FigS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/2/TPJ_4972_sm_TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/3/TPJ_4972_sm_FigS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/4/TPJ_4972_sm_TableS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/5/TPJ_4972_sm_FigS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/6/j.1365-313X.2012.04972.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/7/TPJ_4972_sm_FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/8/TPJ_4972_sm_TableS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/9/TPJ_4972_sm_FigS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/10/TPJ_4972_sm_TableS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/11/TPJ_4972_sm_FigS4.pd

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144600/1/tpj13955_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144600/2/tpj13955.pd

Distinguishing Calliphorid Puparia Through Application of Confocal Laser Scanning Microscopy

The Calliphoridae, or blow flies, are of much ecological and practical importance given their roles in decompositional ecology, medical and veterinary myiasis, and forensic entomology. As ephemeral and rapidly developing species, adults are frequently not present for identification, but puparia, the remaining outer integument of the third instar larvae, frequently are found. These heavily sclerotized remains are stable in the environment, but are character conservative. Historically, scanning electron microscopy (SEM) has been used for characterization, a technique which is not only time consuming but often expensive, effectively making large numbers of specimens impossible to quantify. As an alternative, confocal laser scanning laser microscopy (CSLM) was tested for utility in providing superior data over SEM. Furthermore, due the use of intrinsic autofluorescence for imagining, CSLM is significantly more rapid than SEM requiring no preparation for imaging. Three channels of excitation and emission spectra provided not only image data from the pupal wall, but also from the hydrocarbons found upon the puparia. The laser excitation wavelengths were: 404.7, 488, 640.5 nm and the emissions were: 425–475, 500–550, 663–738 nm. For ten species of Calliphorids, CSLM was used to image puparia. Not only did this provide characters for species identification, but also allowed for the examination of hundreds of specimens. The data generated from the maximum intensity projection images was then used for multivariate discriminate analysis to test for characters useful in delimiting taxa. This provides a basis for puparia identification regardless of image methodology

An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2

Chloroplasts adapt to freezing and other abiotic stresses in part by modifying their membranes. One key-remodeling enzyme is SENSITIVE TO FREEZING2 (SFR2). SFR2 is unusual because it does not respond to initial cold stress or cold acclimation, instead it responds during freezing conditions in Arabidopsis. This response has been shown to be sensitive to cytosolic acidification. The unique lipid products of SFR2 have also been detected in response to non-freezing stresses, but what causes SFR2 to respond in these stresses is unknown. Here, we investigate protoplast isolation as a representative of wounding stress. We show that SFR2 oligogalactolipid products accumulate during protoplast isolation. Notably, we show that protoplast cytosol is acidified during isolation. Modification of the buffers reduces oligogalactolipid accumulation, while prolonged incubation in the isolated state increases it. We conclude that SFR2 activation during protoplast isolation correlates with cytosolic acidification, implying that all SFR2 activation may be dependent on cytosolic acidification. We also conclude that protoplasts can be more gently isolated, reducing their stress

\u3ci\u3eWheat streak mosaic virus\u3c/i\u3e Coat Protein Deletion Mutants Elicit More Severe Symptoms Than Wild-Type Virus in Multiple Cereal Hosts

Previously, we reported that coat protein (CP) of Wheat streak mosaic virus (WSMV) (genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. In this study, we demonstrated that WSMV mutants with deletion of CP amino acids 58 to 84 but not of 36 to 57 induced severe chlorotic streaks and spots, followed by acute chlorosis in wheat, maize, barley, and rye compared with mild to moderate chlorotic streaks and mosaic symptoms by wild-type virus. Deletion of CP amino acids 58 to 84 from the WSMV genome accelerated cell-to-cell movement, with increased accumulation of genomic RNAs and CP, compared with the wildtype virus. Microscopic examination of wheat tissues infected by green fluorescent protein–tagged mutants revealed that infection by mutants lacking CP amino acids 58 to 84 caused degradation of chloroplasts, resulting in acute macroscopic chlorosis. The profile of CP-specific proteins was altered in wheat infected by mutants causing acute chlorosis, compared with mutants eliciting wildtype symptoms. All deletion mutants accumulated CP-specific major protein similarly to that in wild-type virus; however, mutants that elicit acute chlorosis failed to accumulate a 31-kDa minor protein compared with wild-type virus or mutants lacking amino acids 36 to 57. Taken together, these data suggest that deletion of CP amino acids 58 to 84 from the WSMV genome enhanced accumulation of CP and genomic RNA, altered CPspecific protein profiles, and caused severe symptom phenotypes in multiple cereal hosts

The \u3ci\u3ePseudomonas syringae\u3c/i\u3e type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9

Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity

In Vivo and In Vitro Anaerobic Mating in \u3ci\u3eCandida albicans\u3c/i\u3e

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains