Role of Foliar Biointerface Properties and Nanomaterial Chemistry in Controlling Cu Transfer into Wild-Type and Mutant Arabidopsis thaliana Leaf Tissue.

Seven Arabidopsis thaliana mutants with differences in cuticle thickness and stomatal density were foliar exposed to 50 mg L-1 Cu3(PO4)2 nanosheets (NS), CuO NS, CuO nanoparticles, and CuSO4. Three separate fractions of Cu (surface-attached, cuticle, interior leaf) were isolated from the leaf at 0.25, 2, 4, and 8 h. Cu transfer from the surface through the cuticle and into the leaf varied with mutant and particle type. The Cu content on the surface decreased significantly over 8 h but increased in the cuticle. Cu derived from the ionic form had the greatest cuticle concentration, suggesting greater difficulty in moving across this barrier and into the leaf. Leaf Cu in the increased-stomatal mutants was 8.5-44.9% greater than the decreased stomatal mutants, and abscisic acid to close the stomata decreased Cu in the leaf. This demonstrates the importance of nanomaterial entry through the stomata and enables the optimization of materials for nanoenabled agriculture.

Expression of the Type III Secretion System Genes in Epiphytic Erwinia amylovora Cells on Apple Stigmas Benefits Endophytic Infection at the Hypanthium.

Erwinia amylovora causes fire blight on rosaceous plants. One of the major entry points of E. amylovora into hosts is flowers, where E. amylovora proliferates epiphytically on stigmatic and hypanthium surfaces and, subsequently, causes endophytic infection at the hypanthium. The type III secretion system (T3SS) is an important virulence factor in E. amylovora. Although the role of T3SS during endophytic infection is well characterized, its expression during epiphytic colonization and role in the subsequent infection is less understood. Here, we investigated T3SS gene expression in epiphytic E. amylovora on stigma and hypanthium of apple flowers under different relative humidities (RH). On stigma surfaces, T3SS was expressed in a high percentage of E. amylovora cells, and its expression promoted epiphytic growth. On hypanthium surfaces, however, T3SS was expressed in fewer E. amylovora cells than on the stigma, and displayed no correlation with epiphytic growth, even though T3SS expression is essential for infection. E. amylovora cells grown on stigmatic surfaces and then flushed down to the hypanthium displayed a higher level of T3SS expression than cells grown on the hypanthium surface alone. Furthermore, E. amylovora cells precultured on stigma had a higher potential to infect flowers than E. amylovora cells precultured in a T3SS-repressive medium. This suggests that T3SS induction during the stigmatic epiphytic colonization may be beneficial for subsequent infection. Finally, epiphytic expression of T3SS was influenced by RH. Higher percentage of stigmatic E. amylovora cells expressed T3SS under high RH than under low RH.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Striatibotrys neoeucylindrosporus sp. nov., a Stachybotrys-like fungus from North America.

Two isolates from Canada and the USA (UAMH 7122 and UAMH 7211, respectively) previously identified as Stachybotrys eucylindrosporus were studied by morphology and six-locus phylogeny (cmdA, ITS, LSU, rpb2, tef1α and tub2). UAMH 7122 and UAMH 7211 are morphologically related but phylogenetically distinct from Striatibotrys eucylindrosporus (≡Stachybotrys eucylindrosporus) and Str. rhabdosporus. Hence, UAMH 7122 and UAMH 7211 are described as a new species, Striatibotrys neoeucylindrosporus sp. nov. with UAMH 7211 as the holotype. The characters of this species include some phialides proliferating by holoblastic extension of phialides and conidia clavate, subcylindrical or cylindrical ellipsoid, or dumbbell-shaped, dark brown to olivaceous grey when mature, longitudinally striate, 10.3-12.3×3-3.8 µm. A key to the species of Striatibotrys is provided.

Functional characterization of the adenine transporter EaAdeP from the fire blight pathogen Erwinia amylovora and its effect on disease establishment in apples and pears.

Erwinia amylovora is the causal agent of fire blight, an economically important disease of apples and pears. As part of the infection process, Er. amylovora propagates on different plant tissues each with distinct nutrient environments. Here, the biochemical properties of the Er. amylovora adenine permease (EaAdeP) are investigated. Heterologous expression of EaAdeP in nucleobase transporter-deficient Escherichia coli strains, coupled with radiolabel uptake studies, revealed that EaAdeP is a high affinity adenine transporter with a Km of 0.43 ± 0.09 µM. Both Es. coli and Er. amylovora carrying extra copies of EaAdeP are sensitive to growth on the toxic analog 8-azaadenine. EaAdeP is expressed during immature pear fruit infection. Immature pear and apple fruit virulence assays reveal that an E. amylovora ΔadeP::Camr mutant is still able to cause disease symptoms, however, with growth at a lower level, indicating that external adenine is utilized in disease establishment.

An Erwinia amylovora uracil transporter mutant retains virulence on immature apple and pear fruit.

Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. A previous study revealed that an E. amylovora uracil auxotroph was still virulent and can cause disease, suggesting that uracil can be obtained from the host environment. The E. amylovora genome contains a locus encoding for a uracil transporter belonging to the nucleobase cation symporter 2 family, displaying a high level of amino acid sequence similarity to the Escherichia coli UraA. Expression of E. amylovora UraA in nucleobase transporter-deficient E. coli strains, coupled with radiolabeled uptake studies reveal that E. amylovora UraA is a high affinity uracil transporter with a Km of 0.57 µM. Both E. coli and E. amylovora carrying extra copies of E. amylovora UraA are sensitive to growth on the toxic analog 5-fluorouracil. An E. amylovora ΔuraA::Camr mutant is still able to grow and cause disease symptoms on immature pears and apples.

Phytophthora abietivora, A New Species Isolated from Diseased Christmas Trees in Connecticut, U.S.A.

A number of fir species (Abies) are produced as Christmas trees around the world. In particular, Fraser fir (Abies fraseri (Pursh) Poir.) is popular as it yields high-quality Christmas trees in temperate North America and Europe. A Phytophthora sp. causing root rot on Fraser fir was isolated from a Christmas tree farm in Connecticut, U.S.A., and found to be new to science according to morphological and molecular phylogenetic analysis using multilocus DNA sequences from ITS, Cox1, ß-Tub, Nadh1, and Hsp90 loci. Thus, it was described and illustrated as Phytophthora abietivora. An informative Koch's postulates test revealed that P. abietivora was the pathogen causing root rot of Fraser fir.

The solute transport and binding profile of a novel nucleobase cation symporter 2 from the honeybee pathogen Paenibacillus larvae.

Here, we report that a novel nucleobase cation symporter 2 encoded in the genome of the honeybee bacterial pathogen Paenibacillus larvae reveals high levels of amino acid sequence similarity to the Escherichia coli and Bacillus subtilis uric acid and xanthine transporters. This transporter is named P. larvae uric acid permease-like protein (PlUacP). Even though PlUacP displays overall amino acid sequence similarities, has common secondary structures, and shares functional motifs and functionally important amino acids with E. coli xanthine and uric acid transporters, these commonalities are insufficient to assign transport function to PlUacP. The solute transport and binding profile of PlUacP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUacP transports the purines adenine and guanine and the pyrimidine uracil. Hypoxanthine, xanthine, and cytosine are not transported by PlUacP, but, along with uric acid, bind in a competitive manner. PlUacP has strong affinity for adenine Km 7.04 ± 0.18 µm, and as with other bacterial and plant NCS2 proteins, PlUacP function is inhibited by the proton disruptor carbonyl cyanide m-chlorophenylhydrazone. The solute transport and binding profile identifies PlUacP as a novel nucleobase transporter.

Functional characterization of the uracil transporter from honeybee pathogen Paenibacillus larvae.

The genome of the Honeybee bacterial pathogen, Paenibacillus larvae, encodes for protein a with substantial amino acid sequence similarity to the canonical Escherichia coli uracil transporter UraA. P. larvae expresses the uracil permease (PlUP) locus, and is sensitive to the presence of the toxic uracil analog 5-fluorouracil under vegetative growth conditions. The solute transport and binding profile of PlUP was determined by radiolabeled uptake experiments via heterologous expression in nucleobase transporter-deficient Saccharomyces cerevisiae strains. PlUP is specific for the transport of uracil and competitively binds xanthine and uric acid. Further biochemical characterization reveals that PlUP has a strong affinity for uracil with a Km 19.5 ±â€¯1.6 µM. Uracil transport is diminished in the presence of the proton disruptor carbonyl cyanide m-chlorophenylhydrazone, but not by the sodium gradient disruptor Ouabain.

The solute transport profile of two Aza-guanine transporters from the Honey bee pathogen Paenibacillus larvae.

Two nucleobase transporters encoded in the genome of the Honey bee bacterial pathogen Paenibacillus larvae belong to the azaguanine-like transporters and are referred to as PlAzg1 and PlAzg2. PlAzg1 and 2 display significant amino acid sequence similarity, and share predicted secondary structures and functional sequence motifs with two Escherichia coli nucleobase cation symporter 2 (NCS2) members: adenine permease (EcAdeP) and guanine-hypoxanthine permease EcGhxP. However, similarity does not define function. Heterologous complementation and functional analysis using nucleobase transporter-deficient Saccharomyces cerevisiae strains revealed that PlAzg1 transports adenine, hypoxanthine, xanthine and uracil, while PlAzg2 transports adenine, guanine, hypoxanthine, xanthine, cytosine and uracil. Both PlAzg1 and 2 display high affinity for adenine with Km of 2.95 ± 0.22 and 1.92 ± 0.22 µM, respectively. These broad nucleobase transport profiles are in stark contrast to the narrow transport range observed for EcAdeP (adenine) and EcGhxP (guanine and hypoxanthine). PlAzg1 and 2 are similar to eukaryotic Azg-like transporters in that they share a broad solute transport profile, particularly the fungal Aspergillus nidulans AzgA (that transports adenine, guanine and hypoxanthine) and plant AzgA transporters from Arabidopsis thaliana and Zea mays (that collectively move adenine, guanine, hypoxanthine, xanthine, cytosine and uracil).

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations ('Widely Prevalent' clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.

Phylogenetic relationships of Chlamydomyces, Harzia, Olpitrichum, and their sexual allies, Melanospora and Sphaerodes.

Phylogenetic analyses using internal transcribed spacer (ITS), large subunit rRNA (LSU), and small subunit (SSU) sequence data showed that Harzia, Chlamydomyces, and Olpitrichum are con-generic. Thus, Chlamydomyces, and Olpitrichum were reduced to synonymy of Harzia. The generic concept was amended and expanded accordingly. Eight new combinations were proposed. Melanospora and Sphaerodes are phylogenetically related to Harzia. However, several members of Melanospora and Sphaerodes are polyphyletic and belong to Hypocreales or Microascales in Sordariomycetes. The Proteophiala morph is not only a crucial morphological character, but also has a phylogenetical significance in defining Melanosporales. It is hypothesized that the taxa with synanamorphic or asexual Proteophiala all belong to Ceratostomataceae, Melanosporales.

Evidence for a Role for NAD(P)H Dehydrogenase in Concentration of CO2 in the Bundle Sheath Cell of Zea mays.

Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2 This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199-224].

Heterologous complementation studies reveal the solute transport profiles of a two-member nucleobase cation symporter 1 (NCS1) family in Physcomitrella patens.

As part of an evolution-function analysis, two nucleobase cation symporter 1 (NCS1) from the moss Physcomitrella patens (PpNCS1A and PpNCS1B) are examined--the first such analysis of nucleobase transporters from early land plants. The solute specificity profiles for the moss NCS1 were determined through heterologous expression, growth and radiolabeled uptake experiments in NCS1-deficient Saccharomyces cerevisiae. Both PpNCS1A and 1B, share the same profiles as high affinity transporters of adenine and transport uracil, guanine, 8-azaguanine, 8-azaadenine, cytosine, 5-fluorocytosine, hypoxanthine, and xanthine. Despite sharing the same solute specificity profile, PpNCS1A and PpNCS1B move nucleobase compounds with different efficiencies. The broad nucleobase transport profile of PpNCS1A and 1B differs from the recently-characterized Viridiplantae NCS1 in breadth, revealing a flexibility in solute interactions with NCS1 across plant evolution.

The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility.

The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

Light-harvesting complex B7 shifts the irradiance response of photosynthetic light-harvesting regulation in leaves of Arabidopsis thaliana.

The nuclear LHCB7 gene is common in higher plants, encodes a transcript that is well expressed in a subset of leaf mesophyll cells, and is associated with a protein product that is homologous to pigment-binding components of the photosystem (PS) II peripheral antenna complex. We compared the physiological properties of wild type and LHCB7-deficient leaves [DNA insertion, Arabidopsis thaliana (At) ecotype Columbia] in terms of pigment content, CO2 exchange, in vivo transmittance at 810 nm, and chlorophyll fluorescence. The latter two techniques are functional indicators for PSI and PSII, respectively. Key features of the mutant phenotype were confirmed using antisense technology and a hemizygote of two independent AtLHCB7 DNA insertion lines. Growth, leaf pigment composition, white light absorptance, and levels of AtLHCB1-6 were not significantly different in the mutant compared to wild type. Likewise, neither intrinsic PSII light capture efficiency nor partitioning of absorbed radiation to PSII was affected by the mutation. The absence of AtLHCB7 is associated with lower rates of light-saturated photosynthesis and a diminished irradiance threshold for induction of photoprotective non-photochemical quenching. Overall, the pattern of change in light utilization parameters and plastoquinol level indicated that loss of AtLHCB7 expression led to slower Rubisco turnover characterized by pH-dependent balancing of electron transport to reduced carbon assimilation capacity (photosynthetic control). No effect of AtLHCB7 genotype on xanthophyll de-epoxidation state was detected suggesting that factors in addition to lumenal pH influence zeaxanthin accumulation.

The nucleobase cation symporter 1 of Chlamydomonas reinhardtii and that of the evolutionarily distant Arabidopsis thaliana display parallel function and establish a plant-specific solute transport profile.

The single cell alga Chlamydomonas reinhardtii is capable of importing purines as nitrogen sources. An analysis of the annotated C. reinhardtii genome reveals at least three distinct gene families encoding for known nucleobase transporters. In this study the solute transport and binding properties for the lone C. reinhardtii nucleobase cation symporter 1 (CrNCS1) are determined through heterologous expression in Saccharomyces cerevisiae. CrNCS1 acts as a transporter of adenine, guanine, uracil and allantoin, sharing similar - but not identical - solute recognition specificity with the evolutionary distant NCS1 from Arabidopsis thaliana. The results suggest that the solute specificity for plant NCS1 occurred early in plant evolution and are distinct from solute transport specificities of single cell fungal NCS1 proteins.

Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis.

Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis.

In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.

High glycolate oxidase activity is required for survival of maize in normal air.

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.