Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction

Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two‐spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector‐like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage‐specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant‐eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.     

Herbivore performance and plant defense after sequential attacks by inducing and suppressing herbivores

It is well known that herbivore-induced plant defenses alter host plant quality and can affect the behavior and performance of later arriving herbivores.Effects of sequential attacks by herbivores that either suppress or induce plant defenses are less well studied.We sequentially infested leaves of tomato plants with a strain of the phytophagous spider mite Tetranychus urticae that induces plant defenses and the closely related Tetranychus evansi, which suppresses plant defenses.Plant quality was quantified through oviposifion of both spider mite species and by measuring proteinase inhibitor activity using plant material that had been sequentially attacked by both herbivore species.Spider-mite oviposifion data show that T.evansi could suppress an earlier induction of plant defenses by T.urticae,and T.urticae could induce defenses in plants previously attacked by T.evansi in 1 day.Longer attacks by the second species did not result in further changes in oviposifion.Proteinase inhibitor activity levels showed that T.evansi suppressed the high activity levels induced by T.urticae to constitutive levels in 1 day,and further suppressed activity to levels similar to those in plants attacked by T.evansi alone.Attacks by T.urticae induced proteinase inhibitor activity in plants previously attacked by T.evansi,eventually to similar levels as induced by T.urticae alone.Hence,plant quality and plant defenses were significantly affected by sequential attacks and the order of attack does not affect subsequent performance,but does affect proteinase inhibitor activity levels.Based on our results,we discuss the evolution of suppression of plant defenses.     

The distribution of herbivores between leaves matches their performance only in the absence of competitors

Few studies have tested how plant quality and the presence of competitors interact in determining how herbivores choose between different leaves within a plant. We investigated this in two herbivorous spider mites sharing tomato plants: Tetranychus urticae, which generally induces plant defenses, and Tetranychus evansi, which suppresses them, creating asymmetrical effects on coinfesting competitors. On uninfested plants, both herbivore species preferred young leaves, coinciding with increased mite performance. On plants with heterospecifics, the mites did not prefer leaves on which they had a better performance. In particular, T. urticae avoided leaves infested with T. evansi, which is in agreement with T. urticae being outcompeted by T. evansi. In contrast, T. evansi did not avoid leaves with the other species, but distributed itself evenly over plants infested with heterospecifics. We hypothesize that this behavior of T. evansi may prevent further spread of T. urticae over the shared plant. Our results indicate that leaf age determines within‐plant distribution of herbivores only in absence of competitors. Moreover, they show that this distribution depends on the order of arrival of competitors and on their effects on each other, with herbivores showing differences in behavior within the plant as a possible response to the outcome of those interactions.     

Insight into watery saliva proteomes of the grain aphid,Sitobion avenae

The grain aphid, Sitobion avenae, is an economically important cereal pest worldwide. Aphid saliva plays an essential role in the interaction between aphids and their host plants. However, limited information is available regarding the proteins found in the saliva of S. avenae. Here, the watery saliva proteins from S. avenae were collected in an artificial diet and identified using a liquid chromatography–mass spectrometry/mass spectrometry analysis. A total of 114 proteins were identified in S. avenae saliva, including several enzymes, binding proteins, and putative effectors, as well as other proteins with unknown functions. In comparison with salivary proteins from nine other aphid species, the most striking feature of the salivary protein from S. avenae was the different patterns of protein functions. Several orthologous proteins secreted by other aphid species such as glucose dehydrogenase, elongation factors, and effector C002 were also detected in S. avenae saliva and speculated to play a significant role in aphid–plant interactions. These results provide further insight into the molecular basis between aphids and cereal plant interactions.     

Trypanosoma evansi: Paraflagellar rod protein 1 and 2 are similar but lack common B cell epitopes

In an attempt to identify invariant proteins with vaccine potential against African trypanosomes, we investigated the existence of PFR1 protein in Trypanosoma evansi and compared its B cell epitope with that of PFR2 protein of T. evansi using Western blotting and immuno-precipitation assays. The PFR1 gene of T. evansi was amplified by RT-PCR using primers designed based on the open reading frame of PFR1 gene of Trypanosoma brucei. The cloned PFR1 gene of T.evansi was similar to PFR1 genes of T. brucei and Trypanosoma cruzi. The expressed protein from the PFR1 gene was 68.4% homologous to the PFR2 protein of T. evansi, and showed 99.8%, 87%, 77.9% and 77.5% homologous to the PFR1 protein of T. brucei, T. cruzi, Leishmania mexicana and Leishmania major, respectively. Western blot and immuno-precipitation assays showed that antibodies raised against PFR1 and 2 proteins in BALB/c mice recognized the PFR1 and 2 proteins, respectively, with no cross-reactivity. Immuno-agglutination assay showed trypanolytic properties of the anti-PFR1, anti-PFR2 and anti-native PFR sera. These results suggest that PFR1 and PFR2 proteins are components of native PFR antigen and do not share common B cell epitopes.     

Tetranychus evansi spider mite populations suppress tomato defenses to varying degrees

Plant defense suppression is an offensive strategy of herbivores, in which they manipulate plant physiological processes to increase their performance. Paradoxically, defense suppression does not always benefit the defense‐suppressing herbivores, because lowered plant defenses can also enhance the performance of competing herbivores and can expose herbivores to increased predation. Suppression of plant defense may therefore entail considerable ecological costs depending on the presence of competitors and natural enemies in a community. Hence, we hypothesize that the optimal magnitude of suppression differs among locations. To investigate this, we studied defense suppression across populations of Tetranychus evansi spider mites, a herbivore from South America that is an invasive pest of solanaceous plants including cultivated tomato, Solanum lycopersicum, in other parts of the world. We measured the level of expression of defense marker genes in tomato plants after infestation with mites from eleven different T. evansi populations. These populations were chosen across a range of native (South American) and non‐native (other continents) environments and from different host plant species. We found significant variation at three out of four defense marker genes, demonstrating that T. evansi populations suppress jasmonic acid‐ and salicylic acid‐dependent plant signaling pathways to varying degrees. While we found no indication that this variation in defense suppression was explained by differences in host plant species, invasive populations tended to suppress plant defense to a smaller extent than native populations. This may reflect either the genetic lineage of T. evansi—as all invasive populations we studied belong to one linage and both native populations to another—or the absence of specialized natural enemies in invasive T. evansi populations.     

Characterization of the Proteome of Theobroma cacao Beans by Nano‐UHPLC‐ESI MS/MS

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano‐UHPLC‐ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species‐specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non‐specific databases confirmed that using a species‐specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586.     

Protein profile of Lupinus texensis phloem sap exudates: Searching for Fe‐ and Zn‐containing proteins

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI‐MS and ESI‐MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19–21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe‐containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe‐binding proteins in phloem sap: a metallothionein‐like protein type 2B identified in the Fe‐affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem‐specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn‐binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.     

Trypanosoma brucei brucei: A comparison of gene expression in the liver and spleen of infected mice utilizing cDNA microarray technology

Trypanosoma brucei brucei, the infectious agent of the disease known as Nagana, is a pathogenic trypanosome occurring in Africa, where it causes significant economic loss to domesticated livestock. Although many studies on the histopathology of organs of mice infected with T. b. brucei have been reported, little work has been done regarding gene expression in these organs in infected mice. In this paper, we describe the use of cDNA microarray to determine gene expression profiles in the liver and spleen of mice infected with T. b. brucei (STIB 920) at peak parasitaemia (12 days after infection). Our results showed that a total of 123 genes in the liver and 389 genes in the spleen were expressed differentially in T. b. brucei infected mice. In contrast, however, in an acute infection in mice caused by Trypanosoma brucei evansi, a species genetically related to T. b. brucei, 336 genes in the liver and 190 genes in the spleen were expressed, differentially, indicating that the liver of mice was more affected by the acute T. b. evansi infection whilst the spleen was more affected by the subacute T. b. brucei infection. Our results provide a number of possible reasons why mice infected with T. b. evansi die sooner than those infected with T. b. brucei: (1) mice infected with T. b. evansi may need more stress response proteins to help them pass through the infection and these are probably excessively consumed; (2) proliferating cell nuclear antigen was more down-regulated in the liver of mice infected with T. b. evansi, which indicated that the inhibition of proliferation of hepatocytes in mice infected with T. b. evansi might be more severe than that in T. b. brucei infection; and (3) more hepatocyte apoptosis occurred in the mice infected with T. b. evansi and this might be probably the most important reason why mice died sooner than those infected with T. b. brucei. Studies of the changes in the gene expression profile in the liver and spleen of mice infected with T. b. brucei may be helpful in understanding the mechanisms of pathogenesis in Nagana disease at the molecular level. By comparing the gene profiles of the liver and spleen of mice infected with T. b. brucei with T. b. evansi, we have identified a number of factors that could explain the differences in pathogenesis in mice infected with these two African trypanosomes.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

Life history of the predatory mite <Emphasis Type="Italic">Phytoseiulus fragariae</Emphasis> on <Emphasis Type="Italic">Tetranychus evansi</Emphasis> and <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Phytoseiidae,Tetranychidae) at five temperatures

Tetranychus evansi Baker and Pritchard and Tetranychus urticae Koch (Acari: Tetranychidae) are important pests of Solanaceae in many countries. Several studies have demonstrated that T. urticae is an acceptable prey to many predatory mites, although the suitability of this prey depends on the host plant. T. evansi, has been shown to be an unfavorable prey to most predatory mites that have been tested against it. The predator Phytoseiulus fragariae Denmark and Schicha (Acari: Phytoseiidae) has been found in association with the two species in Brazil. The objective of this work was to compare biological parameters of P. fragariae on T. evansi and on T. urticae as prey. The study was conducted under laboratory conditions at 10, 15, 20, 25 and 30°C. At all temperatures, survivorship was lower on T. evansi than on T. urticae. No predator reached adulthood at 10°C on the former species; even on the latter species, only about 36% of the predators reached adulthood at 10°C. For both prey, in general, duration of each life stage was shorter, total fecundity was lower and intrinsic rate of population increase (r _m ) was higher with increasing temperatures. The slower rate of development of P. fragariae on T. evansi resulted in a slightly higher thermal requirement (103.9 degree-days) on that prey than on T. urticae (97.1 degree-days). The values of net reproduction rate (R ₀), intrinsic rate of increase (r _m ) and finite rate of increase (λ) were significantly higher on T. urticae, indicating faster population increase of the predator on this prey species. The highest value of r _m of the predator was 0.154 and 0.337 female per female per day on T. evansi and on T. urticae, respectively. The results suggested that P. fragariae cannot be considered a good predator of T. evansi.     

The invasive spider mite Tetranychus evansi (Acari: Tetranychidae) alters community composition and host-plant use of native relatives

The tomato spider mite Tetranychus evansi Baker and Pritchard (Acari: Tetranychidae), is a worldwide pest of solanaceous crops that has recently invaded many parts of the world. In the present study we examined the ecological impact of its arrival in the Mediterranean region. The spider mite and phytoseiid mite assemblages in various crop and non-crop plants in three areas of Valencia (Spain) were studied a few months before and 10 years after the invasion of T. evansi. According to rarefaction analyses, the invasion of T. evansi did not affect neither the total number of species in the mite community examined (spider mite and phytoseiid species) nor the number of species when the two communities were examined separately. However, after the invasion, the absolute and relative abundance of the native Tetranychus species was significantly reduced. Before the invasion, T. urticae and T. turkestani were the most abundant spider mites, accounting for 62.9 and 22.8 % of the specimens. After the invasion, T. evansi became the most abundant species, representing 60 % of the total spider mites recorded, whereas the abundance of T. urticae was significantly reduced (23 %). This reduction took place principally on non-crop plants, where native species were replaced by the invader. Null model analyses provided evidence for competition structuring the spider mite community on non-crop plants after the invasion of T. evansi. Resistance to acaricides, the absence of efficient native natural enemies, manipulation of the plant defenses and the web type produced by T. evansi are discussed as possible causes for the competitive displacement.     

Search for effective natural enemies of Tetranychus evansi in south and southeast Brazil

Tetranychus evansi Baker & Pritchard is an important pest of Solanaceae in several countries. Introduced accidentally to Africa, it presently occurs in many countries of that continent. In some of them, it is considered a key pest. The suspected area of origin of this mite is South America. The objective of the present study was to identify phytoseiid mites on solanaceous plants in association with T. evansi in south and southeast Brazil for introduction in the African continent for use in a classic biological control program. Almost 1,400 predatory mites of the family Phytoseiidae were collected, on 22 solanaceous species. The Amblyseiinae were the most diverse group in this study. Twenty-three of the species found belong to this subfamily, while only three belong to the Typhlodrominae and two to the Phytoseiinae. The most abundant and most frequent phytoseiid species were Phytoseius guianensis De Leon and Galendromus annectens (De Leon) of the Phytoseiinae and Typhlodrominae, respectively. The most frequent and abundant species of Amblyseiinae was Neoseiulus tunus (De Leon). Phytoseius guianensis and N. tunus were never found in association with T. evansi and G. annectens was found only once in association with it. Two factors suggested Phytoseiulus longipes Evans as the most promising predator found in this study. It could walk very well on tomato leaves infested by T. evansi, without being hampered by the profuse webbing produced by the prey and by the trichomes. In addition, several specimens of both sexes including eggs and nymphs of the predator were found associated with T. evansi on three different plant species and in two different periods of the year, when T. evansi was the only arthropod present on the leaves.     

Comparative proteomic analysis of the saliva of the Rhodnius prolixus,Triatoma lecticularia and Panstrongylus herreri triatomines reveals a high interespecific functional biodiversity

Triatomines are hematophagous arthropods that transmit Trypanosoma cruzi and Trypanosoma rangeli. Feeding behavior and pathogen transmission is known to vary between the different species, and this characteristic is directly or indirectly dependent on the bioactive molecules of the saliva that facilitate the vector–host–parasite interaction. Here, we identify, characterize and compare the sialoproteomic (from the Greek sialo: saliva) repertoire of important species of the main triatomine genera in the Americas (Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri) to better explain this interaction through two-dimensional electrophoresis and mass spectrometry. We identified 221 proteins, 69 from R. prolixus, 100 from T. lecticularia and 52 from P. herreri. We identified high abundance molecules with a great potential to modulate host defenses and homeostasis, highlighting Nitrophorin-4 (28.7%), Salivary lipocalin-5 (65.2%) and Putative triabin (20.5%) in R. prolixus, T. lecticularia and P. herreri, respectively. We also observed that only a single hypothetical protein is shared among three species, which was not functionally categorized. This study corroborates previous findings with R. prolixus, increasing the knowledge about this species with relevant proteomic information and comparisons with the other two targets of the study, T. lecticularia and P. herreri, for which no studies are available from a proteomics perspective.     

Cloning and characterization of MS5 from Arabidopsis : a gene critical in male meiosis

In this paper, we describe the cloning of the MS5 gene, a gene essential for male fertility in Arabidopsis . We previously defined the MS5 locus by characterizing an EMS-induced allele, ms5–1 . We identified a new allele of MS5 ( ms5–2 ) that was T-DNA-generated and used the T-DNA tag to clone the gene. Sequencing of mutant and wild-type alleles together with complementation of the ms5–1 mutant phenotype with a wild-type genomic clone confirmed the identity of the gene. Differences between the phenotypes of the two mutant alleles could be attributed to differences in mutant gene structure. The semi-dominant and dominant negative phenotypes of the ms5–2 mutant probably result from production of a truncated polypeptide. An unknown locus in Landsberg erecta can counteract the dominant negative phenotype of ms5–2 . Mutations in MS5 cause the formation of ‘polyads’– tetrads with more than four pools of chromosomes after male meiosis. Similarities between the MS5 sequence and that of a number of proteins were found; two that may be significant were with a synaptonemal complex protein and with a regulatory subunit of a cyclin-dependent kinase. The MS5 gene is a member of a small gene family highly conserved amongst plant species.     

Proteins Identified from Saliva and Salivary Glands of the Chinese Gall Aphid Schlechtendalia chinensis

Aphid saliva plays an essential role in the interaction between aphids and their host plants. Several aphid salivary proteins have been identified but none from galling aphids. Here the salivary proteins from the Chinese gall aphid are analyzed, Schlechtendalia chinensis, via an LC‐MS/MS analysis. A total of 31 proteins are identified directly from saliva collected via an artificial diet, and 141 proteins are identified from extracts derived from dissected salivary glands. Among these identified proteins, 17 are found in both collected saliva and dissected salivary glands. In comparison with salivary proteins from ten other free‐living Hemipterans, the most striking feature of the salivary protein from S. chinensis is the existence of high proportion of proteins with binding activity, including DNA‐, protein‐, ATP‐, and iron‐binding proteins. These proteins maybe involved in gall formation. These results provide a framework for future research to elucidate the molecular basis for gall induction by galling aphids.     

Life-history of predatory mites Neoseiulus californicus and Phytoseiulus persimilis (Acari: Phytoseiidae) on four spider mite species as prey, with special reference to Tetranychus evansi (Acari: Tetranychidae)

The commercially available strains of Phytoseiulus persimilis Athias-Henriot, the biological control agent of Tetranychus urticae Koch, perform poorly in the Western Mediterranean, probably because they are not well adapted to local climatic conditions. For that reason, efforts are being focused on the development of a biological control programme using native phytoseiid mites. Four species of red spider mites can be found in vegetable crops in eastern Spain: T. urticae, Tetranychus turkestani Ugarov and Nikolski, Tetranychus ludeni Zacher and the recently introduced Tetranychus evansi Baker and Pritchard. To evaluate their potential role as biological control agents, the present study evaluates the life-history of local populations of Neoseiulus californicus (McGregor) and P. persimilis when fed on T. urticae, T. turkestani, T. evansi, and T. ludeni in the laboratory. Results indicate that N. californicus and P. persimilis are able to feed and complete their development on the four tested red spider mite species. The predators may exhibit a particularly high capacity for population increase when fed on T. urticae, T. turkestani, and T. ludeni, thus may be able to provide effective control of these species in the field. When fed T. evansi, however, predator performance was poor; significant increase in development and preoviposition times, and a reduction in oviposition period and fecundity were recorded. The resultant low capacity for population growth suggests poor ability of the two tested predators to suppress T. evansi populations on commercial crops. It is unlikely therefore that P. persimilis and N. californicus, now being widely used to control T. urticae in greenhouse crops in Central Europe, will be able to halt any spread of T. evansi to greenhouse crops in temperate areas.     

Comparative analyses of three legume species reveals conserved and unique root extracellular proteins

Increasing evidence suggests that root extracellular proteins are involved in interactions between roots and their soil environment. In the present study, exudates released by 6‐day‐old roots of the three legume species white lupin (Lupinus albus), soybean (Glycine max), and cowpea (Vigna sinensis) were collected under axenic conditions, and their constitutively secreted proteomes were analyzed. Between 42 and 93 unique root extracellular proteins with 2 or more different peptide fragments per protein were identified by LC‐MS/MS. Functional annotation of these proteins classified them into 14–16 different functional categories. Among those 14 homologous proteins were identified in at least two legume species. Among the unique proteins, 58 in white lupin, 85 in soybean, and 31 in cowpea were specific for each plant species, and many of them were classified in the same functional categories. Interestingly, in contrast to soybean and cowpea, two protein bands of approximately 16 and 30 kDa were present on the SDS‐PAGE gel of white lupin. The identification of these bands revealed a class III chitinase and a thaumatin‐like protein. Both belong to the class of pathogenesis‐related proteins. The results imply that root extracellular proteins play important roles in the cross‐talk between plant roots and the rhizosphere.