SfREPAT38, a pathogen response gene (REPAT), is involved in immune response of Spodoptera frugiperda larvae through mediating Toll signalling pathway.

REPAT (response to pathogen) is an immune-associated gene family that plays important roles in insect immune response to pathogens. Although nine REPAT genes have been identified in Spodoptera frugiperda (Lepidoptera: Noctuidae) currently, their functions and mechanisms in the immune response to pathogens still remain unclear. Therefore, SfREPAT38, a pathogen response gene (REPAT) of S. frugiperda, was characterised and its function was analysed. The results showed that SfREPAT38 contains a signal peptide and a transcription activator MBF2 (multi-protein bridging factor 2) domain. Quantitative real-time polymerase chain reaction analysis showed that SfREPAT38 was highly expressed in the sixth-instar larvae (L6) and was the highest in expression in the midgut of L6. We found that the expression of SfREPAT38 could be activated by challenge with four microbial pathogens (Bacillus thuringiensis, Metarhizium anisopliae, Spodoptera exigua nuclearpolyhedrosis and Escherichia coli), except 12 h after E. coli infection. Furthermore, the SfREPAT38 expression levels significantly decreased at 24, 48 and 72 h after SfREPAT38 dsRNA injection or feeding. Feeding with SfREPAT38 dsRNA significantly decreased the weight gain of S. frugiperda, and continuous feeding led to the death of S. frugiperda larvae from the fourth day. Moreover, SfREPAT38 dsRNA injection resulted in a significant decrease of weight gain on the fifth day. Silencing SfREPAT38 gene down-regulated the expression levels of immune genes belonging to the Toll pathway, including SPZ, Myd88, DIF, Cactus, Pell and Toll18W. After treatment with SfREPAT38 dsRNA, S. frugiperda became extremely sensitive to the B. thuringiensis infection, and the survival rate dramatically increased, with 100% mortality by the eighth day. The weight of S. frugiperda larvae was also significantly lower than that of the control groups from the second day onwards. In addition, the genes involved in the Toll signalling pathway and a few antibacterial peptide related genes were down-regulated after treatment. These results showed that SfREPAT38 is involved in the immune response of S. frugiperda larvae through mediating Toll signalling pathway.

GhMYB18 confers Aphis gossypii Glover resistance through regulating the synthesis of salicylic acid and flavonoids in cotton plants.

KEY MESSAGE: R2R3 MYB transcription factor GhMYB18 is involved in the defense response to cotton aphid by participating in the synthesis of salicylic acid and flavonoids. R2R3 MYB transcription factors (TFs) play crucial roles in plant growth and development as well as response to abiotic and biotic stresses. However, the mechanism of R2R3 MYB TFs in cotton response to aphid infestation remains largely unknown. Here, an R2R3 MYB transcription factor GhMYB18 was identified as a gene up-regulated from upland cotton (Gossypium hirsutum L.) under cotton aphid (Aphis gossypii Glover) infestation. GhMYB18, which has transcription activity, was localized mainly to nucleus and cell membranes. Transient overexpression of GhMYB18 in cotton activates salicylic acid (SA) and phenylpropane signaling pathways and promoted the synthesis of salicylic acid and flavonoids, which leads to enhancing the tolerance to cotton aphid feeding. In contrast, silencing of GhMYB18 increased the susceptibility of G. hirsutum to aphid. Additionally, GhMYB18 significantly promoted the activities of defense-related enzymes including catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL). These results collectively suggest that GhMYB18 is involved in cotton defense response to cotton aphid attacks through regulating the synthesis of salicylic acid and flavonoids.

Sensing Home: A Cost-Effective Design for Smart Home via Heterogeneous Wireless Networks.

The aging population has inspired the marketing of advanced real time devices for home health care, more and more wearable devices and mobile applications, which have emerged in this field. However, to properly collect behavior information, accurately recognize human activities, and deploy the whole system in a real living environment is a challenging task. In this paper, we propose a feasible wireless-based solution to deploy a data collection scheme, activity recognition model, feedback control and mobile integration via heterogeneous networks. We compared and found a suitable algorithm that can be run on cost-efficient embedded devices. Specifically, we use the Super Set Transformation method to map the raw data into a sparse binary matrix. Furthermore, designed front-end devices of low power consumption gather the living data of the habitant via ZigBee to reduce the burden of wiring work. Finally, we evaluated our approach and show it can achieve a theoretical time-slice accuracy of 98%. The mapping solution we propose is compatible with more wearable devices and mobile apps.

Photoreactivation Activities of Rad5, Rad16A and Rad16B Help Beauveria bassiana to Recover from Solar Ultraviolet Damage.

In budding yeast, Rad5 and Rad7-Rad16 play respective roles in the error-free post-replication repair and nucleotide excision repair of ultraviolet-induced DNA damage; however, their homologs have not yet been studied in non-yeast fungi. In the fungus Beauveria bassiana, a deficiency in the Rad7 homolog, Rad5 ortholog and two Rad16 paralogs (Rad16A/B) instituted an ability to help the insect-pathogenic fungus to recover from solar UVB damage through photoreactivation. The fungal lifecycle-related phenotypes were not altered in the absence of rad5, rad16A or rad16B, while severe defects in growth and conidiation were caused by the double deletion of rad16A and rad16B. Compared with the wild-type and complemented strains, the mutants showed differentially reduced activities regarding the resilience of UVB-impaired conidia at 25 °C through a 12-h incubation in a regime of visible light plus dark (L/D 3:9 h or 5:7 h for photoreactivation) or of full darkness (dark reactivation) mimicking a natural nighttime. The estimates of the median lethal UVB dose LD50 from the dark and L/D treatments revealed greater activities of Rad5 and Rad16B than of Rad16A and additive activities of Rad16A and Rad16B in either NER-dependent dark reactivation or photorepair-dependent photoreactivation. However, their dark reactivation activities were limited to recovering low UVB dose-impaired conidia but were unable to recover conidia impaired by sublethal and lethal UVB doses as did their photoreactivation activities at L/D 3:9 or 5:7, unless the night/dark time was doubled or further prolonged. Therefore, the anti-UV effects of Rad5, Rad16A and Rad16B in B. bassiana depend primarily on photoreactivation and are mechanistically distinct from those for their yeast homologs.

High photoreactivation activities of Rad2 and Rad14 in recovering insecticidal Beauveria bassiana from solar UV damage.

Anti-ultraviolet (UV) roles of Rad2 and Rad14 depend on nucleotide excision repair (NER) of UV-induced DNA lesions in budding yeast but remain unexplored yet in filamentous fungi. Here, nucleus-specific Rad2 and Rad14 orthologs are shown to recover Beauveria bassiana, a main source of wide-spectrum mycoinsecticides, from solar UV damage through photorepair-depending photoreactivation. As a photorepair index, photoreactivation (germination) rates of lethal UVB dose-irradiated conidia via a 3- or 5-h light plus 9- or 7-h dark incubation at 25 °C were drastically reduced in the Δrad2 and Δrad14 mutants versus a wild-type strain. As an NER index, nighttime-mimicking 12-h dark reactivation rates of low UVB dose-impaired conidia decreased sharply compared to the corresponding photoreactivation rates in the presence or absence of either ortholog, indicating that its extant NER activity was limited to recovering light UVB damage in the field. The high photoreactivation activity of either Rad2 or Rad14 was derived from its tight link to a large protein complex formed by photolyase regulators and other anti-UV proteins through multiple protein-protein interactions revealed by yeast two-hybrid assays. Therefore, Rad2 and Rad14 recover B. bassiana from solar UV damage through photoreactiovation in vivo that depends primarily on photorepair, although they contribute little to the fungal lifecycle-related phenotypes. These findings unveil a novel scenario distinguished from the NER-depending anti-UV roles of Rad2 and Rad14 in the model yeast and broaden a biological basis crucial for rational application of fungal insecticides to improve pest control efficacy via feasible recovery of solar UV damage.

Rad6, a ubiquitin conjugator required for insect-pathogenic lifestyle, UV damage repair, and genomic expression of Beauveria bassiana.

The E2 ubiquitin conjugator Rad6 is required for DNA damage bypass in budding yeast but remain functionally unknown in filamentous fungi. Here, we report pleiotropic effect of Rad6 ortholog in Beauveria bassiana, a wide-spectrum fungal insecticide. Global ubiquitination signal was greatly attenuated in the absence of rad6. The blocked ubiquitination led to severe growth defect, blocked asexual development, and abolished infectivity/insect pathogenicity, which correlated with compromised conidial quality (including viability, hydrophobicity, adherence to insect cuticle, and thermotolerance) and blocked secretion of cuticle-degrading enzymes including Pr1 family proteases. Importantly, Rad6 played much greater role in photoreactivation of UVB-impaired conidia by a 3- or 5-h light plus 9- or 7-h dark incubation than in dark reactivation of those impaired conidia by a 12-h dark incubation. The high activity of Rad6 in photoreactivation in vivo was derived from its link to a protein complex cored by the photolyase regulators WC1 and WC2 via the strong interactions of Rad6 with the E3 partner Rad18 and Rad18 with WC2 revealed in yeast two-hybrid assays. Transcriptomic analysis resulted in identification of 2700 differentially regulated genes involved in various function categories and metabolism pathways, indicating a regulatory role of Rad6-mediated ubiquitination in gene expression networks and genomic stability. Conclusively, Rad6 is required for asexual and insect-pathogenic lifecycles, solar UV damage repair, and genomic expression of B. bassiana. The primary dependence of its strong anti-UV role on photoreactivation in vivo unveils a scenario distinct from the core role of its yeast ortholog in DNA damage bypass.

Comparative Roles of Rad4A and Rad4B in Photoprotection of Beauveria bassiana from Solar Ultraviolet Damage.

The Rad4-Rad23-Rad33 complex plays an essential anti-ultraviolet (UV) role depending on nucleotide excision repair (NER) in budding yeast but has been rarely studied in filamentous fungi, which possess two Rad4 paralogs (Rad4A/B) and orthologous Rad23 and rely on the photorepair of UV-induced DNA lesions, a distinct mechanism behind the photoreactivation of UV-impaired cells. Previously, nucleocytoplasmic shuttling Rad23 proved to be highly efficient in the photoreactivation of conidia inactivated by UVB, a major component of solar UV, due to its interaction with Phr2 in Beauveria bassiana, a wide-spectrum insect mycopathogen lacking Rad33. Here, either Rad4A or Rad4B was proven to localize exclusively in the nucleus and interact with Rad23, which was previously shown to interact with the white collar protein WC2 as a regulator of two photorepair-required photolyases (Phr1 and Phr2) in B. bassiana. The Δrad4A mutant lost ~80% of conidial UVB resistance and ~50% of activity in the photoreactivation of UVB-inactivated conidia by 5 h light exposure. Intriguingly, the reactivation rates of UVB-impaired conidia were observable only in the presence of rad4A after dark incubation exceeding 24 h, implicating extant, but infeasible, NER activity for Rad4A in the field where night (dark) time is too short. Aside from its strong anti-UVB role, Rad4A played no other role in B. bassiana's lifecycle while Rad4B proved to be functionally redundant. Our findings uncover that the anti-UVB role of Rad4A depends on the photoreactivation activity ascribed to its interaction with Rad23 linked to WC2 and Phr2 and expands a molecular basis underlying filamentous fungal adaptation to solar UV irradiation on the Earth's surface.

Co-Regulatory Roles of WC1 and WC2 in Asexual Development and Photoreactivation of Beauveria bassiana.

The white collar proteins WC1 and WC2 interact with each other to form a white collar complex acting as a well-known transcription regulator required for the operation of the circadian clock in Neurospora, but their roles in insect-pathogenic fungal lifecycles remain poorly understood. Here, we report that WC1 and WC2 orthologs co-regulate the conidiation capacity and conidial resistance to solar ultraviolet-B (UVB) irradiation in Beauveria bassiana, after their high activities in the photorepair of UVB-induced DNA damages were elucidated previously in the insect mycopathogen, which features non-rhythmic conidiation and high conidiation capacity. The conidial yield, UVB resistance, and photoreactivation rate of UVB-impaired conidia were greatly reduced in the null mutants of wc1 and wc2 compared to their control strains. However, many other lifecycle-related phenotypes, except the antioxidant response, were rarely affected in the two mutants. Transcriptomic analysis revealed largely overlapping roles for WC1 and WC2 in regulating the fungal gene networks. Most of the differentially expressed genes identified from the null mutants of wc1 (1380) and wc2 (1001) were co-downregulated (536) or co-upregulated (256) at similar levels, including several co-downregulated genes required for aerial conidiation and DNA photorepair. These findings expand a molecular basis underlying the fungal adaptation to solar UV irradiation and offer a novel insight into the genome-wide co-regulatory roles of WC1 and WC2 in B. bassiana's asexual development and in vivo photoreactivation against solar UV damage.

GhMYC1374 regulates the cotton defense response to cotton aphids by mediating the production of flavonoids and free gossypol.

Myelocytomatosis (MYC) transcription factors (TFs) in plants are well-known regulators of plant defense against herbivores. However, the role and mechanism of MYC TFs in cotton (Gossypium hirsutum L.) defense against cotton aphids (Aphis gossypii Glover) remain still elusive. Herein, on the basis of aphid-induced cotton transcriptome analysis, GhMYC1374, a cotton MYC2-like TF that was highly induced by cotton aphid attack, has been identified that confers cotton aphid resistance in cotton. GhMYC1374 was an intranuclear transcription factor with three domains: bHLH-MYC_N, RBR and bHLH_AtAIB_like. GhMYC1374 was induced under cotton aphid feeding, exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments. GhMYC1374 transient overexpression in cotton plants enhanced cotton aphid-resistance, while GhMYC1374 silence through VIGS (virus induced gene silencing) decreased cotton aphid-resistance. GhMYC1374 transient overexpression of in cotton plants activated the phenylpropane pathway and promoted the synthesis of flavonoids, and resistance to thus enhanced the cotton resistance against aphids. In contrast, GhMYC1374 silence inhibited the biosynthesis of flavonoids. In addition, GhMYC1374 also positively activated the expression of the biosynthetic genes of free gossypol, leading to the high content of free gossypol. Taken together, our results suggest that GhMYC1374 is involved in the cotton defense response against cotton aphids by regulating the biosynthesis of flavonoids and free gossypol.

Differential Roles of Five Fluffy Genes (flbA-flbE) in the Lifecycle In Vitro and In Vivo of the Insect-Pathogenic Fungus Beauveria bassiana.

The fluffy genes flbA-flbE are well-known players in the upstream developmental activation pathway that activates the key gene brlA of central developmental pathway (CDP) to initiate conidiation in Aspergillus nidulans. Here, we report insignificant roles of their orthologs in radial growth of Beauveria bassiana under normal culture conditions and different stresses although flbA and flbD were involved in respective responses to heat shock and H2O2. Aerial conidiation level was lowered in the deletion mutants of flbB and flbE (~15%) less than of flbA and flbC (~30%), in which the key CDP genes brlA and abaA were repressed consistently during normal incubation. The CDP-controlled blastospore production in submerged cultures mimicking insect hemolymph was abolished in the flbA mutant with brlA and abaA being sharply repressed, and decreased by 55% in the flbC mutant with only abaA being downregulated. The fungal virulence against a model insect was attenuated in the absence of flbA more than of flbC irrespective of normal cuticle infection or cuticle-bypassing infection (intrahemocoel injection). These findings unravel more important role of flbA than of flbC, but null roles of flbB/D/E, in B. bassiana's insect-pathogenic lifecycle and a scenario distinctive from that in A.nidulans.

Rad1 and Rad10 Tied to Photolyase Regulators Protect Insecticidal Fungal Cells from Solar UV Damage by Photoreactivation.

Beauveria bassiana serves as a main source of global fungal insecticides, which are based on the active ingredient of formulated conidia vulnerable to solar ultraviolet (UV) irradiation and restrained for all-weather application in green agriculture. The anti-UV proteins Rad1 and Rad10 are required for the nucleotide excision repair (NER) of UV-injured DNA in model yeast, but their anti-UV roles remain rarely exploredin filamentous fungi. Here, Rad1 and Rad10 orthologues that accumulated more in the nuclei than the cytoplasm of B. bassiana proved capable of reactivating UVB-impaired or UVB-inactivated conidia efficiently by 5h light exposure but incapable of doing so by 24 h dark incubation (NER) if the accumulated UVB irradiation was lethal. Each orthologue was found interacting with the other and two white collar proteins (WC1 and WC2), which proved to be regulators of two photolyases (Phr1 and Phr2) and individually more efficient in the photorepair of UVB-induced DNA lesions than either photolyase alone. The fungal photoreactivation activity was more or far more compromised when the protein-protein interactions were abolished in the absence of Rad1 or Rad10 than when either Phr1 or Phr2 lost function. The detected protein-protein interactions suggest direct links of either Rad1 or Rad10 to two photolyase regulators. In B. bassiana, therefore, Rad1 and Rad10 tied to the photolyase regulators have high activities in the photoprotection of formulated conidia from solar UV damage but insufficient NER activities in the field, where night (dark) time is too short, and no other roles in the fungal lifecycle in vitro and in vivo.

FluG and FluG-like FlrA Coregulate Manifold Gene Sets Vital for Fungal Insect-Pathogenic Lifestyle but Not Involved in Asexual Development.

The central developmental pathway (CDP) activator gene brlA is activated by the upstream genes fluG and flbA-flbE in Aspergillus nidulans. Increasing evidences of fungal genome divergence make it necessary to clarify whether such genetic principles fit Pezizomycotina. Previously, fluG disruption resulted in limited conidiation defect and little effect on the expression of brlA and flbA-flbE in Beauveria bassiana possessing the other FluG-like regulator FlrA. Here, single-disruption (SD) mutants of flrA and double-disruption (DD) mutants of flrA and fluG were analyzed to clarify whether FlrA and FluG are upstream regulators of key CDP genes. Despite similar subcellular localization, no protein-protein interaction was detected between FlrA and FluG, suggesting mutual independence. Three flrA SD mutants showed phenotypes similar to those previously described for ΔfluG, including limited conidiation defect, facilitated blastospore production, impaired spore quality, blocked host infection, delayed proliferation in vivo, attenuated virulence, and increased sensitivities to multiple stresses. Three DD mutants resembled the SD mutants in all phenotypes except more compromised pathogenicity and tolerance to heat shock- or calcofluor white-induced stress. No CDP gene appeared in 1,622 and 2,234 genes dysregulated in the ΔflrA and ΔfluG mutants, respectively. The majority (up/down ratio: 540:875) of those dysregulated genes were co-upregulated or co-downregulated at similar levels in the two mutants. These findings unravel novel roles for flrA and fluG in coregulating manifold gene sets vital for fungal adaptation to insect-pathogenic lifestyle and environment but not involved in CDP activation. IMPORTANCE FluG is a core regulator upstream of central developmental pathway (CDP) in Aspergillus nidulans but multiple FluG-like regulators (FLRs) remain functionally uncharacterized in ascomycetes. Our previous study revealed no role for FluG in the CDP activation and an existence of sole FLR (FlrA) in an insect-pathogenic fungus. This study reveals a similarity of FlrA to FluG in domain architecture and subcellular localization. Experimental data from analyses of targeted single- and double-gene knockout mutants demonstrate similar roles of FrlA and FluG in stress tolerance and infection cycle but no role of either in CDP activation. Transcriptomic analyses reveal that FlrA and FluG coregulate a large number of same genes at similar levels. However, the regulated genes include no key CDP gene. These findings uncover that FlrA and FluG play similar roles in the fungal adaptation to insect-pathogenic lifestyle and environment but no role in the activation of CDP.

GhCalS5 is involved in cotton response to aphid attack through mediating callose formation.

Callose synthase plays an essential role in plant growth and development and in response to all sorts of stresses through regulating callose formation. However, few research about the function and mechanism of the insect resistance of callose synthase genes have been reported in cotton. In this study, a cotton callose synthase gene GhCalS5 was cloned, and its function and mechanism of resistance to cotton aphids were analyzed. The expression of GhCalS5 was significantly upregulated in both, leaves and stems of cotton plants at 48 h after cotton aphid infestation and in the leaves of cotton plants at 24 h after salicylic acid treatment. The overexpression of GhCalS5 enhanced cotton resistance to cotton aphids. Expectedly silencing of GhCalS5 reduced cotton resistance to cotton aphids. Overexpression of GhCalS5 enhanced callose formation in cotton leaves. Our results suggest that GhCalS5 is involved in cotton resistance against cotton aphids by influencing callose formation.

ZmRop1 participates in maize defense response to the damage of Spodoptera frugiperda larvae through mediating ROS and soluble phenol production.

As plant-specific molecular switches, Rho-like GTPases (Rops) are vital for plant survival in response to biotic and abiotic stresses. However, their roles in plant defense response to phytophagous insect's damage are largely unknown. In this study, the expression levels of nine maize RAC family genes were analyzed after fall armyworm (FAW) larvae infestation. Among the analyzed genes, ZmRop1 was specifically and highly expressed, and its role in maize response to FAW larvae damage was studied. The results showed that upon FAW larvae infestation, salicylic acid and methyl jasmonate treatment ZmRop1 gene transcripts were all down-regulated. However, upon mechanical injury, the expression level of ZmRop1 was up-regulated. Overexpression of ZmRop1 gene in maize plants could improve maize plant resistance to FAW larvae damage. Conversely, silencing of ZmRop1 increased maize plant susceptibility to FAW larvae damage. The analysis of the potential anti-herbivore metabolites, showed that ZmRop1 promoted the enzyme activities of catalase, peroxidase and the expression levels of ZmCAT, ZmPOD, ZmRBOHA and ZmRBOHB, thereby enhancing the reactive oxygen species (ROS) production, including the content of O2- and H2O2. In addition, overexpression or silencing of ZmRop1 could have influence on the content of the total soluble phenol through mediating the activity of polyphenol oxidase. In summary, the results illuminated our understanding of how ZmRop1 participate in maize defense response to FAW larvae damage as a positive regulator through mediating ROS production and can be used as a reference for the green prevention and control of FAW larvae.