The role of a cytosolic superoxide dismutase in barley–pathogen interactions

Reactive oxygen species (ROS), including superoxide ( / ) and hydrogen peroxide (H₂O₂), are differentially produced during resistance responses to biotrophic pathogens and during susceptible responses to necrotrophic and hemi‐biotrophic pathogens. Superoxide dismutase (SOD) is responsible for the catalysis of the dismutation of / to H₂O₂, regulating the redox status of plant cells. Increased SOD activity has been correlated previously with resistance in barley to the hemi‐biotrophic pathogen Pyrenophora teres f. teres (Ptt, the causal agent of the net form of net blotch disease), but the role of individual isoforms of SOD has not been studied. A cytosolic CuZnSOD, HvCSD1, was isolated from barley and characterized as being expressed in tissue from different developmental stages. HvCSD1 was up‐regulated during the interaction with Ptt and to a greater extent during the resistance response. Net blotch disease symptoms and fungal growth were not as pronounced in transgenic HvCSD1 knockdown lines in a susceptible background (cv. Golden Promise), when compared with wild‐type plants, suggesting that cytosolic / contributes to the signalling required to induce a defence response to Ptt. There was no effect of HvCSD1 knockdown on infection by the hemi‐biotrophic rice blast pathogen Magnaporthe oryzae or the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei, but HvCSD1 also played a role in the regulation of lesion development by methyl viologen. Together, these results suggest that HvCSD1 could be important in the maintenance of the cytosolic redox status and in the differential regulation of responses to pathogens with different lifestyles.     

The lre-miR159a-LrGAMYB pathway mediates resistance to grey mould infection in Lilium regale

Grey mould is one of the most determinative factors of lily growth and plays a major role in limiting lily productivity. MicroRNA159 (miR159) is a highly conserved microRNA in plants, and participates in the regulation of plant development and stress responses. Our previous studies revealed that lre-miR159a participates in the response of Lilium regale to Botrytis elliptica according to deep sequencing analyses; however, the response mechanism remains unknown. Here, lre-miR159a and its target LrGAMYB gene were isolated from L. regale. Transgenic Arabidopsis overexpressing lre-MIR159a exhibited larger leaves and smaller necrotic spots on inoculation with Botrytis than those of wild-type and overexpressing LrGAMYB plants. The lre-MIR159a overexpression also led to repressed expression of two targets of miR159, AtMYB33 and AtMYB65, and enhanced accumulation of hormone-related genes, including AtPR1, AtPR2, AtNPR1, AtPDF1.2, and AtLOX for both the jasmonic acid and salicylic acid pathways. Moreover, lower levels of H₂O₂ and were observed in lre-MIR159a transgenic Arabidopsis, which reduced the damage from reactive oxygen species accumulation. Taken together, these results indicate that lre-miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response.     

Verticillium dahliae secreted protein Vd424Y is required for full virulence,targets the nucleus of plant cells,and induces cell death

Fungal pathogens secrete effector proteins that regulate host immunity and can suppress basal defence mechanisms against colonization in plants. Verticillium dahliae is a widespread and destructive soilborne fungus that can cause vascular wilt disease and reduces plant yields. However, little is currently known about how the effectors secreted by V. dahliae function. In this study, we analysed and identified 34 candidate effectors in the V. dahliae secretome and found that Vd424Y, a glycoside hydrolase family 11 protein, was highly upregulated during the early stages of V. dahliae infection in cotton plants. This protein was located in the nucleus and its deletion compromised the virulence of the fungus. The transient expression of Vd424Y in Nicotiana benthamiana induced BAK1- and SOBIR1-dependent cell death and activated both salicylic acid and jasmonic acid signalling. This enhanced its resistance to the oomycetes Phytophthora capsici in a way that depended on its nuclear localization signal and signal peptides. Our results demonstrate that Vd424Y is an important effector protein targeting the host nucleus to regulate and activate effector-triggered immunity in plants.     

The glycoside hydrolase 28 member VdEPG1 is a virulence factor of Verticillium dahliae and interacts with the jasmonic acid pathway-related gene GhOPR9

Glycoside hydrolase (GH) family members act as virulence factors and regulate plant immune responses during pathogen infection. Here, we characterized the GH28 family member endopolygalacturonase VdEPG1 in Verticillium dahliae. VdEPG1 acts as a virulence factor during V. dahliae infection. The expression level of VdEPG1 was greatly increased in V. dahliae inoculated on cotton roots. VdEPG1 suppressed VdNLP1-mediated cell death by modulating pathogenesis-related genes in Nicotiana benthamiana. Knocking out VdEPG1 led to a significant decrease in the pathogenicity of V. dahliae in cotton. The deletion strains were more susceptible to osmotic stress and the ability of V. dahliae to utilize carbon sources was deficient. In addition, the deletion strains lost the ability to penetrate cellophane membrane, with mycelia showing a disordered arrangement on the membrane, and spore development was affected. A jasmonic acid (JA) pathway-related gene, GhOPR9, was identified as interacting with VdEPG1 in the yeast two-hybrid system. The interaction was further confirmed by bimolecular fluorescence complementation and luciferase complementation imaging assays in N. benthamiana leaves. GhOPR9 plays a positive role in the resistance of cotton to V. dahliae by regulating JA biosynthesis. These results indicate that VdEPG1 may be able to regulate host immune responses as a virulence factor through modulating the GhOPR9-mediated JA biosynthesis.     

Plant defence suppression is mediated by a fungal sirtuin during rice infection by Magnaporthe oryzae

Crop destruction by the hemibiotrophic rice pathogen Magnaporthe oryzae requires plant defence suppression to facilitate extensive biotrophic growth in host cells before the onset of necrosis. How this is achieved at the genetic level is not well understood. Here, we report that a M. oryzae sirtuin, MoSir2, plays an essential role in rice defence suppression and colonization by controlling superoxide dismutase (SOD) gene expression. Loss of MoSir2 function in Δsir2 strains did not affect appressorial function, but biotrophic growth in rice cells was attenuated. Compared to wild type, Δsir2 strains failed to neutralize plant‐derived reactive oxygen species (ROS) and elicited robust defence responses in rice epidermal cells that included elevated pathogenesis‐related gene expression and granular depositions. Deletion of a SOD‐encoding gene under MoSir2 control generated Δsod1 deletion strains that mimicked Δsir2 for impaired rice defence suppression, confirming SOD activity as a downstream output of MoSir2. In addition, comparative protein acetylation studies and forward genetic analyses identified a JmjC domain‐containing protein as a likely target of MoSir2, and a Δsir2 Δjmjc double mutant was restored for MoSOD1 expression and defence suppression in rice epidermal cells. Together, this work reveals MoSir2 and MoJmjC as novel regulators of early rice cell infection.     

The Verticillium dahliae Sho1-MAPK pathway regulates melanin biosynthesis and is required for cotton infection

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.     

An estimator of the Opportunity for Selection that is independent of mean fitness

Variation among individuals in number of offspring (fitness, k) sets an upper limit to the evolutionary response to selection. This constraint is quantified by Crow's Opportunity for Selection (I), which is the variance in relative fitness (I = σ²_k/(u_k)²). Crow's I has been widely used but remains controversial because it depends on mean offspring number in a sample (). Here, I used a generalized Wright-Fisher model that allows for unequal probabilities of producing offspring to evaluate behavior of Crow's I and related indices under a wide range of sampling scenarios. Analytical and numerical results are congruent and show that rescaling the sample variance (s²_k) to its expected value at a fixed removes dependence of I on mean offspring number, but the result still depends on choice of . A new index is introduced, Δ_I = Π– E(Î_drift) = Π– 1/, which makes Î independent of sample without the need for variance rescaling. Δ_I has a straightforward interpretation as the component of variance in relative fitness that exceeds that expected under a null model of random reproductive success. Δ_I can be used to directly compare estimates of the Opportunity for Selection for samples from different studies, different sexes, and different life stages.     

Comparative effects of ammonium,nitrate and urea on growth and photosynthetic efficiency of three bloom‐forming cyanobacteria

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GhADF6-mediated actin reorganization is associated with defence against Verticillium dahliae infection in cotton

Several studies have revealed that actin depolymerizing factors (ADFs) participate in plant defence responses; however, the functional mechanisms appear intricate and need further exploration. In this study, we identified an ADF6 gene in upland cotton (designated as GhADF6) that is evidently involved in cotton's response to the fungal pathogen Verticillium dahliae. GhADF6 binds to actin filaments and possesses actin severing and depolymerizing activities in vitro and in vivo. When cotton root (the site of the fungus invasion) was inoculated with the pathogen, the expression of GhADF6 was markedly down-regulated in the epidermal cells. By virus-induced gene silencing analysis, the down-regulation of GhADF6 expression rendered the cotton plants tolerant to V. dahliae infection. Accordingly, the abundance of actin filaments and bundles in the root cells was significantly higher than that in the control plant, which phenocopied that of the V. dahliae-challenged wild-type cotton plant. Altogether, our results provide evidence that an increase in filament actin (F-actin) abundance as well as dynamic actin remodelling are required for plant defence against the invading pathogen, which are likely to be fulfilled by the coordinated expressional regulation of the actin-binding proteins, including ADF.     

Infrared-to-visible conversion in strontium sulphate through a defect-based infrared stimulated visible emission phenomenon

Strontium sulphate (SrSO₄) is a defect-based photoluminescence material, generally used in thermoluminescence applications, and has been studied for infrared (IR) stimulated visible emission. The SrSO₄ particles were synthesized using a precipitation method. The orthorhombic phase of SrSO₄ was confirmed from the X-ray diffraction pattern and the formation of micron-sized particles was authenticated from field emission scanning electron micrographs. The elemental composition of oxygen and strontium was determined using energy-dispersive X-ray analysis measurement that confirmed the presence of $V_O^{••}$ and $V_{\mathit{Sr}}^{\prime \prime }$ intrinsic defects in the material. Photoluminescence investigations showed the presence of various defect bands in the band gap giving rise to intrinsic luminescence in SrSO₄. The emission in the visible region was attributed to the defect band arising due to $V_O^{••}$. Photoluminescence lifetime measurement confirmed the presence of stable defect states with a lifetime in microseconds. The SrSO₄ sample was tested using IR lasers and a red–orange emission spot was observed from the powder sample when excited with IR lasers. The underlying principle for IR-to-visible conversion in the material is a defect-mediated phenomenon that has been described through the energy level diagram of the material.     

Characterization of a Novel Cotton Subtilase Gene GbSBT1 in Response to Extracellular Stimulations and Its Role in Verticillium Resistance

Verticillium wilt is a disastrous vascular disease in plants caused by Verticillium dahliae. Verticillium pathogens secrete various disease-causing effectors in cotton. This study identified a subtilase gene GbSBT1 from Gossypium babardense and investigated the roles against V. dahliae infection. GbSBT1 gene expression is responsive to V. dahliae defense signals, jasmonic acid, and ethylene treatments. Moreover, the GbSBT1 protein is mainly localized in the cell membrane and moves into the cytoplasm following jasmonic acid and ethylene treatments. Silencing GbSBT1 gene expression through virus-induced GbSBT1 gene silencing reduced the tolerance of Pima-90 (resistant genotype), but not facilitated the infection process of V. dahliae in Coker-312 (sensitive genotype). Moreover, the ectopically expressed GbSBT1 gene enhanced the resistance of Arabidopsis to Fusarium oxysporum and V. dahliae infection and activated the expression levels of defense-related genes. Furthermore, pull-down, yeast two-hybrid assay, and BiFC analysis revealed that GbSBT1 interacts with a prohibitin (PHB)-like protein expressed in V. dahliae pathogens during infection. In summary, GbSBT1 recognizes the effector PHB protein secreted from V. dahliae and is involved in Verticillium-induced resistance in cotton.     

High throughput screening of ultrafiltration and diafiltration processing of monoclonal antibodies via the ambr® crossflow system

As the biopharmaceutical industry moves toward high concentration of monoclonal antibody drug substance, additional development is required early on when material is still limited. A key constraint is the availability of predictive high-throughput low-volume filtration screening systems for bioprocess development. This particularly impacts final stages such as ultrafiltration/diafiltration steps where traditional scale-down systems need hundreds of milliliters of material per run. Recently, the ambr® crossflow system has been commercialized by Sartorius Stedim Biotech (SSB) to meet this need. It enables parallel high throughput experimentation by only using a fraction of typical material requirements. Critical parameters for predictive filtration systems include loading, mean transmembrane pressure (Δ$\overline{P}$ _TMP), and crossflow rate (Q_F). While axial pressure drop (ΔP_axial) across the cartridge is a function of these parameters, it plays a key role and similar values should result across scales. The ambr® crossflow system is first presented describing typical screening experiments. Its performance is then compared to a traditional pilot-scale tangential flow filtration (TFF) at defined conditions. The original ambr® crossflow (CF) cartridge underperformed resulting in ~20x lower ΔP_axial than the pilot-scale TFF flat-sheet cassette. With an objective to improve the scalability of the system, efforts were made to understand this scale difference. The ambr® CF cartridge was successfully modified by restricting the flow of the feed channel, and thus increasing its ΔP_axial. Additional studies across a range of loading (100–823 gm⁻²); Δ$\overline{P}$ _TMP (12–18 psi); and Q_F (4–8 L/min/m²) were conducted in both scales. Comparable flux and aggregate levels were achieved.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

Use of thermal imaging and the photochemical reflectance index (PRI) to detect wheat response to elevated CO2 and drought

The spectral-based photochemical reflectance index (PRI) and leaf surface temperature (T_leaf) derived from thermal imaging are two indicative metrics of plant functioning. The relationship of PRI with radiation-use efficiency (RUE) and T_leaf with leaf transpiration could be leveraged to monitor crop photosynthesis and water use from space. Yet, it is unclear how such relationships will change under future high carbon dioxide concentrations ([CO₂]) and drought. Here we established an [CO₂] enrichment experiment in which three wheat genotypes were grown at ambient (400 ppm) and elevated (550 ppm) [CO₂] and exposed to well-watered and drought conditions in two glasshouse rooms in two replicates. Leaf transpiration (T_r) and latent heat flux (LE) were derived to assess evaporative cooling, and RUE was calculated from assimilation and radiation measurements on several dates along the season. Simultaneous hyperspectral and thermal images were taken at $~$1.5 m from the plants to derive PRI and the temperature difference between the leaf and its surrounding air ($∆$T_leaf−air). We found significant PRI and RUE and $∆$T_leaf−air and T_r correlations, with no significant differences among the genotypes. A PRI–RUE decoupling was observed under drought at ambient [CO₂] but not at elevated [CO₂], likely due to changes in photorespiration. For a LE range of 350 W m^–2, the ΔT_leaf−air range was $~$10°C at ambient [CO₂] and only $~$4°C at elevated [CO₂]. Thicker leaves in plants grown at elevated [CO₂] suggest higher leaf water content and consequently more efficient thermoregulation at high [CO₂] conditions. In general, T_leaf was maintained closer to the ambient temperature at elevated [CO₂], even under drought. PRI, RUE, ΔT_leaf−air, and T_r decreased linearly with canopy depth, displaying a single PRI-RUE and ΔT_leaf−air T_r model through the canopy layers. Our study shows the utility of these sensing metrics in detecting wheat responses to future environmental changes.     

The mitochondrial membrane protein FgLetm1 regulates mitochondrial integrity,production of endogenous reactive oxygen species and mycotoxin biosynthesis in Fusarium graminearum

Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS‐generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper‐EF‐hand‐containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial‐produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.     

Defence-related Enzymes in Pepper Roots During Interactions with Arbuscular Mycorrhizal Fungi and/or Verticillium dahliae

Previous studies have described that arbuscular mycorrhizal fungi (AMF) can reduce the deleterious effect of Verticillium dahliae Kleb. on pepper growth and yield. In mycorrhizal plants, the bioprotection against soil-borne pathogens can result from the preactivation of defence responses that include some structural modifications and the accumulation of Pathogenesis-Related (PR) proteins. Our first objective was to study if V. dahliae induced defence mechanisms in roots before infected pepper developed visible symptoms of disease. The second aim was to determine if AMF induced defence-related enzymatic activities in pepper roots before or after pathogen’s attack. Results showed that the colonization of pepper roots by Glomus deserticola (Trappe, Bloss and Menge) induced the appearance of new isoforms of acidic chitinases, superoxide dismutase (SOD) and, at early stages, peroxidases. In contrast, V. dahliae neither stimulated the phenylpropanoid pathway nor elicited hydrolytic activities in infected pepper roots. Only in mycorrhizal plants, the inoculation with V. dahliae slightly increased both phenylalanine ammonia-lyase (PAL) and peroxidase activities two weeks later. Mycorrhizal-specific induction of new isoforms of acidic chitinases and SOD together with enhanced peroxidase and PAL activities 2 weeks after pathogen inoculation could be involved in the biocontrol of Verticillium-induced wilt in pepper by AMF.