The cotton MAPK kinase GhMPK20 negatively regulates resistance to Fusarium oxysporum by mediating the MKK4–MPK20–WRKY40 cascade

Fusarium wilt is one of the most serious diseases affecting cotton. However, the pathogenesis and mechanism by which Fusarium oxysporum overcomes plant defence responses are unclear. Here, a new group D mitogen‐activated protein kinase (MAPK) gene, GhMPK20, was identified and functionally analysed in cotton. GhMPK20 expression was significantly induced by F. oxysporum. Virus‐induced gene silencing (VIGS) of GhMPK20 in cotton increased the tolerance to F. oxysporum, whereas ectopic GhMPK20 overexpression in Nicotiana benthamiana reduced F. oxysporum resistance via disruption of the salicylic acid (SA)‐mediated defence pathway. More importantly, an F. oxysporum‐induced MAPK cascade pathway composed of GhMKK4, GhMPK20 and GhWRKY40 was identified. VIGS of GhMKK4 and GhWRKY40 also enhanced F. oxysporum resistance in cotton, and the function of GhMKK4–GhMPK20 was shown to be essential for F. oxysporum‐induced GhWRKY40 expression. Together, our results indicate that the GhMKK4–GhMPK20–GhWRKY40 cascade in cotton plays an important role in the pathogenesis of F. oxysporum. This research broadens our knowledge of the negative role of the MAPK cascade in disease resistance in cotton and provides an important scientific basis for the formulation of Fusarium wilt prevention strategies.     

Maternal control of embryogenesis by MPK6 and its upstream MKK4/MKK5 in Arabidopsis

In flowering plants, developing embryos reside in maternal sporophytes. It is known that maternal generation influences the development of next‐generation embryos; however, little is known about the signaling components in the process. Previously, we demonstrated that Arabidopsis mitogen‐activated protein kinase 6 (MPK6) and MPK3 play critical roles in plant reproduction. In addition, we noticed that a large fraction of seeds from mpk6 single‐mutant plants showed a wrinkled seed coat or a burst‐out embryo phenotype. Here, we report that these seed phenotypes can be traced back to defective embryogenesis. The defective embryos have shorter suspensors and reduced growth along the longitudinal axis. Furthermore, the cotyledons fail to bend over to progress to the bent‐cotyledon stage. As a result of the uneven circumference along the axis, the seed coat wrinkles to develop raisin‐like morphology after dehydration. In more severe cases, the embryo can be pushed out from the micropylar end, resulting in the burst‐out embryo seed phenotype. Genetic analyses demonstrated that the defective embryogenesis of the mpk6 mutant is a maternal effect. Heterozygous or homozygous mpk6 embryos have defects only in mpk6 homozygous maternal plants, but not in wild‐type or heterozygous maternal plants. The loss of function of MKK4/MKK5 also results in the same phenotypes, suggesting that MKK4/MKK5 might act upstream of MPK6 in this pathway. The maternal‐mediated embryo defects are associated with changes in auxin activity maxima and PIN localization. In summary, this research demonstrates that the Arabidopsis MKK4/MKK5–MPK6 cascade is an important player in the maternal control of embryogenesis.     

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum)

Cultivated cotton (Gossypium hirsutum) is the most important fibre crop in the world. Cotton leaf curl disease (CLCuD) is the major limiting factor and a threat to textile industry in India and Pakistan. All the local cotton cultivars exhibit moderate to no resistance against CLCuD. In this study, we evaluated an exotic cotton accession Mac7 as a resistance source to CLCuD by challenging it with viruliferous whiteflies and performing qPCR to evaluate the presence/absence and relative titre of CLCuD‐associated geminiviruses/betasatellites. The results indicated that replication of pathogenicity determinant betasatellite is significantly attenuated in Mac7 and probably responsible for resistance phenotype. Afterwards, to decipher the genetic basis of CLCuD resistance in Mac7, we performed RNA sequencing on CLCuD‐infested Mac7 and validated RNA‐Seq data with qPCR on 24 independent genes. We performed co‐expression network and pathway analysis for regulation of geminivirus/betasatellite‐interacting genes. We identified nine novel modules with 52 hubs of highly connected genes in network topology within the co‐expression network. Analysis of these hubs indicated the differential regulation of auxin stimulus and cellular localization pathways in response to CLCuD. We also analysed the differential regulation of geminivirus/betasatellite‐interacting genes in Mac7. We further performed the functional validation of selected candidate genes via virus‐induced gene silencing (VIGS). Finally, we evaluated the genomic context of resistance responsive genes and found that these genes are not specific to A or D sub‐genomes of G. hirsutum. These results have important implications in understanding CLCuD resistance mechanism and developing a durable resistance in cultivated cotton.     

Responses of Thrips tabaci to odours of herbivore‐induced cotton seedlings

Herbivore‐induced changes in plants have been widely viewed as defensive responses against further insect attack. However, changes in plants as a consequence of herbivore feeding can elicit various responses in herbivores; these are variable, context dependent, and often unpredictable. In this laboratory study, the responses of Thrips tabaci Lindeman (Thysanoptera: Thripidae) to volatiles emitted by intact and herbivore‐damaged or mechanically damaged cotton seedlings [Gossypium hirsutum L. (Malvaceae)] were investigated in dual‐choice olfactometer assays. Thrips tabaci showed increased attraction to seedlings subject to foliar mechanical damage and those with foliar damage inflicted by conspecifics or Tetranychus urticae Koch (Acari: Tetranychidae), upon which it preys. However, T. tabaci did not discriminate between intact seedlings and those with foliar damage inflicted by Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), two other species of thrips, Frankliniella schultzei Trybom and Frankliniella occidentalis Pergrande (Thysanoptera: Thripidae), or those with root damage inflicted by Tenebrio molitor L. (Coleoptera: Tenebrionidae). Attraction of T. tabaci was also affected by herbivore density on damaged plants. That is, seedlings damaged by higher densities of T. urticae or T. tabaci were more attractive than seedlings damaged by lower densities of the corresponding arthropod. Although attracted to plants damaged by conspecifics or T. urticae, T. tabaci showed greater attraction to seedlings damaged by T. urticae than to seedlings damaged by conspecifics. Results are discussed in the context of the responses of F. schultzei and F. occidentalis to herbivore‐induced cotton seedlings, highlighting the complexity, variability, and unpredictability of the responses of even closely related species of insects to plants under herbivore attack.     

Beta‐amyloid 1‐42 monomers,but not oligomers,produce PHF‐like conformation of Tau protein

The mechanistic relationship between amyloid β1‐42 (Aβ1‐42) and the alteration of Tau protein are debated. We investigated the effect of Aβ1‐42 monomers and oligomers on Tau, using mice expressing wild‐type human Tau that do not spontaneously develop Tau pathology. After intraventricular injection of Aβ1‐42, mice were sacrificed after 3 h or 4 days. The short‐lasting treatment with Aβ monomers, but not oligomers, showed a conformational PHF‐like change of Tau, together with hyperphosphorylation. The same treatment induced increase in concentration of GSK3 and MAP kinases. The inhibition of the kinases rescued the Tau changes. Aβ monomers increased the levels of total Tau, through the inhibition of proteasomal degradation. Aβ oligomers reproduced all the aforementioned alterations only after 4 days of treatment. It is known that Aβ1‐42 monomers foster synaptic activity. Our results suggest that Aβ monomers physiologically favor Tau activity and dendritic sprouting, whereas their excess causes Tau pathology. Moreover, our study indicates that anti‐Aβ therapies should be targeted to Aβ1‐42 monomers too.     

Stay or move: how Bt‐susceptible Helicoverpa armigera neonates behave on Bt cotton plants

Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae occasionally have been reported to survive at management threshold levels in fields of Bollgard II^® cotton, Gossypium hirsutum L. (Malvaceae). The pattern and degree of larval survival is not easily predicted but depends on the ability of first instars to establish on host plants. Experiments were conducted with Bacillus thuringiensis Berliner (Bt)‐susceptible and Bt‐resistant larvae of H. armigera to understand how physiologically Bt‐susceptible H. armigera survive on Bt cotton plants, and examine how their first meal influences survival rates. In assays using cotton plant parts, both strains of larvae displayed similar tendencies to drop‐off specific plant parts of Bt and non‐Bt cotton. However, significantly more Bt‐susceptible larvae dropped off young leaves, mature leaves, and squares of Bt cotton compared to non‐Bt cotton plants. Egg cannibalism significantly improved the survival of Bt‐susceptible H. armigera larvae on Bt cotton plants. Larvae were more likely to eat live aged eggs, than newly laid or dead eggs. Survival significantly improved when larvae cannibalized eggs before feeding on Bt leaves. The behavior of Bt‐susceptible larvae with respect to drop‐off and egg cannibalism may help enhance their survival on Bt cotton plants.     

Consumptive and non‐consumptive effects of wolf spiders on cotton bollworms

Larvae of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) that survive on genetically modified Bt cotton (Gossypium hirsutum L., Malvaceae) contribute to the risk of widespread resistance to Bt toxins. Current resistance management techniques include pupae busting, which involves deep tilling of the soil to kill overwintering pupae. Unfortunately, pupae busting runs counter to soil and water conserving techniques, such as minimum tillage. This problem could be relieved with biological control methods, whereby predators attack either larvae going to ground to pupate or moths emerging from the ground. We found that the wolf spider Tasmanicosa leuckartii (Thorell) (Araneae: Lycosidae), a common inhabitant of Australian cotton agroecosystems, is an effective predator of H. armigera, attacking and killing most larvae (66%) and emerging moths (77%) in simple laboratory arenas. Tasmanicosa leuckartii also reduced the number of emerging moths by 66% on average in more structurally complex glasshouse arenas. Males, females, and late‐instar juveniles of T. leuckartii were similarly effective. Tasmanicosa leuckartii also imposed non‐consumptive effects on H. armigera, as when a spider was present larvae in the laboratory areas spent less time on the cotton boll and more time on the soil and more mass was lost from the cotton boll. Increased loss of boll mass likely reflects changes in H. armigera foraging behavior induced by the presence of spiders (indirect non‐consumptive effects). Helicoverpa armigera spent more time as pupae when the spider was present in simple laboratory arenas, but not in more complex glasshouse enclosures. Overall, results indicate that T. leuckartii spiders can be effective predators of H. armigera late instars and moths but also suggest that, under some conditions, the presence of spiders could increase the damage to individual cotton bolls.     

Precise spatio‐temporal modulation of ACC synthase by MPK6 cascade mediates the response of rose flowers to rehydration

Drought is a major abiotic stress that affects the development and growth of most plants, and limits crop yield worldwide. Although the response of plants to drought has been well documented, much less is known about how plants respond to the water recovery process, namely rehydration. Here, we describe the spatio‐temporal response of plant reproductive organs to rehydration using rose flowers as an experimental system. We found that rehydration triggered rapid and transient ethylene production in the gynoecia. This ethylene burst serves as a signal to ensure water recovery in flowers, and promotes flower opening by influencing the expression of a set of rehydration‐responsive genes. An in‐gel kinase assay suggested that the rehydration‐induced ethylene burst resulted from transient accumulation of RhACS1/2 proteins in gynoecia. Meanwhile, RhMPK6, a rose homolog of Arabidopsis thaliana MPK6, is rapidly activated by rehydration within 0.5 h. Furthermore, RhMPK6 was able to phosphorylate RhACS1 but not RhACS2 in vitro. Application of the kinase inhibitor K252a suppressed RhACS1 accumulation and rehydration‐induced ethylene production in gynoecia, and the protein phosphatase inhibitor okadaic acid had the opposite effect, confirming that accumulation of RhACS1 was phosphorylation‐dependent. Finally, silencing of RhMPK6 significantly reduced ethylene production in gynoecia when flowers were subjected to rehydration. Taken together, our results suggest that temporal‐ and spatial‐specific activation of an RhMPK6‐RhACS1 cascade is responsible for rehydration‐induced ethylene production in gynoecia, and that the resulting ethylene‐mediated signaling pathway is a key factor in flower rehydration.     

Glucose metabolism down‐regulates the uptake of 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) mediated by glucose transporter 1 isoform (GLUT1): theory and simulations using the symmetric four‐state carrier model

The non‐metabolizable fluorescent glucose analogue 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) is increasingly used to study cellular transport of glucose. Intracellular accumulation of exogenously applied 6‐NBDG is assumed to reflect concurrent gradient‐driven glucose uptake by glucose transporters (GLUTs). Here, theoretical considerations are provided that put this assumption into question. In particular, depending on the microscopic parameters of the carrier proteins, theory proves that changes in glucose transport can be accompanied by opposite changes in flow of 6‐NBDG. Simulations were carried out applying the symmetric four‐state carrier model on the GLUT1 isoform, which is the only isoform whose kinetic parameters are presently available. Results show that cellular 6‐NBDG uptake decreases with increasing rate of glucose utilization under core‐model conditions, supported by literature, namely where the transporter is assumed to work in regime of slow reorientation of the free‐carrier compared with the ligand–carrier complex. To observe an increase of 6‐NBDG uptake with increasing rate of glucose utilization, and thus interpret 6‐NBDG increase as surrogate of glucose uptake, the transporter must be assumed to operate in regime of slow ligand–carrier binding, a condition that is currently not supported by literature. Our findings suggest that the interpretation of data obtained with NBDG derivatives is presently ambiguous and should be cautious because the underlying transport kinetics are not adequately established.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

The tomato I gene for Fusarium wilt resistance encodes an atypical leucine‐rich repeat receptor‐like protein whose function is nevertheless dependent on SOBIR1 and SERK3/BAK1

We have identified the tomato I gene for resistance to the Fusarium wilt fungus Fusarium oxysporum f. sp. lycopersici (Fol) and show that it encodes a membrane‐anchored leucine‐rich repeat receptor‐like protein (LRR‐RLP). Unlike most other LRR‐RLP genes involved in plant defence, the I gene is not a member of a gene cluster and contains introns in its coding sequence. The I gene encodes a loopout domain larger than those in most other LRR‐RLPs, with a distinct composition rich in serine and threonine residues. The I protein also lacks a basic cytosolic domain. Instead, this domain is rich in aromatic residues that could form a second transmembrane domain. The I protein recognises the Fol Avr1 effector protein, but, unlike many other LRR‐RLPs, recognition specificity is determined in the C‐terminal half of the protein by polymorphic amino acid residues in the LRRs just preceding the loopout domain and in the loopout domain itself. Despite these differences, we show that I/Avr1‐dependent necrosis in Nicotiana benthamiana depends on the LRR receptor‐like kinases (RLKs) SERK3/BAK1 and SOBIR1. Sequence comparisons revealed that the I protein and other LRR‐RLPs involved in plant defence all carry residues in their last LRR and C‐terminal LRR capping domain that are conserved with SERK3/BAK1‐interacting residues in the same relative positions in the LRR‐RLKs BRI1 and PSKR1. Tyrosine mutations of two of these conserved residues, Q922 and T925, abolished I/Avr1‐dependent necrosis in N. benthamiana, consistent with similar mutations in BRI1 and PSKR1 preventing their interaction with SERK3/BAK1.     

The island cotton NBS‐LRR gene GbaNA1 confers resistance to the non‐race 1 Verticillium dahliae isolate Vd991

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non‐commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS‐LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus‐induced gene silencing of each of the eight NBS‐LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non‐functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full‐length (～1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non‐race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.