Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

Cucurbit aphid‐borne yellows virus (CABYV) modifies the alighting,settling and probing behaviour of its vector Aphis gossypii favouring its own spread

Plant pathogens are able to influence the behaviour and fitness of their vectors in such a way that changes in plant–pathogen–vector interactions can affect their transmission. Such influence can be direct or indirect, depending on whether it is mediated by the presence of the pathogen in the vector's body or by host changes as a consequence of pathogen infection. We report the effect that the persistently aphid‐transmitted Cucurbit aphid‐borne yellows virus (CABYV, Polerovirus) can induce on the alighting, settling and probing behaviour activities of its vector, the cotton aphid Aphis gossypii. Only minor direct changes on aphid feeding behaviour were observed when viruliferous aphids fed on non‐infected plants. However, the feeding behaviour of non‐viruliferous aphids was very different on CABYV‐infected than on non‐infected plants. Non‐viruliferous aphids spent longer time feeding from the phloem in CABYV‐infected plants compared to non‐infected plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. Viruliferous aphids showed a clear preference for non‐infected over CABYV‐infected plants at short and long time, while such behaviour was not observed for non‐viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimise the transmission and spread of this phloem‐limited virus.     

Abscisic acid deficiency increases defence responses against Myzus persicae in Arabidopsis

Comparison of Arabidopsis thaliana (Arabidopsis) gene expression induced by Myzus persicae (green peach aphid) feeding, aphid saliva infiltration and abscisic acid (ABA) treatment showed a significant positive correlation. In particular, ABA‐regulated genes are over‐represented among genes that are induced by M. persicae saliva infiltration into Arabidopsis leaves. This suggests that the induction of ABA‐related gene expression could be an important component of the Arabidopsis–aphid interaction. Consistent with this hypothesis, M. persicae populations induced ABA production in wild‐type plants. Furthermore, aphid populations were smaller on Arabidopsis aba1‐1 mutants, which cannot synthesize ABA, and showed a significant preference for wild‐type plants compared with the mutant. Total free amino acids, which play an important role in aphid nutrition, were not altered in the aba1‐1 mutant line, but the levels of isoleucine (Ile) and tryptophan (Trp) were differentially affected by aphids in wild‐type and mutant plants. Recently, indole glucosinolates have been shown to promote aphid resistance in Arabidopsis. In this study, 4‐methoxyindol‐3‐ylmethylglucosinolate was more abundant in the aba1‐1 mutant than in wild‐type Arabidopsis, suggesting that the induction of ABA signals that decrease the accumulation of defence compounds may be beneficial for aphids.     

Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust

Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T₃ to T₄ generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.     

Pea aphid promotes amino acid metabolism both in Medicago truncatula and bacteriocytes to favor aphid population growth under elevated CO2

Rising atmospheric CO₂ levels can dilute the nitrogen (N) resource in plant tissue, which is disadvantageous to many herbivorous insects. Aphids appear to be an exception that warrants further study. The effects of elevated CO₂ (750 ppm vs. 390 ppm) were evaluated on N assimilation and transamination by two Medicago truncatula genotypes, a N‐fixing‐deficient mutant (dnf1) and its wild‐type control (Jemalong), with and without pea aphid (Acyrthosiphon pisum) infestation. Elevated CO₂ increased population abundance and feeding efficiency of aphids fed on Jemalong, but reduced those on dnf1. Without aphid infestation, elevated CO₂ increased photosynthetic rate, chlorophyll content, nodule number, biomass, and pod number for Jemalong, but only increased pod number and chlorophyll content for dnf1. Furthermore, aphid infested Jemalong plants had enhanced activities of N assimilation‐related enzymes (glutamine synthetase, Glutamate synthase) and transamination‐related enzymes (glutamate oxalate transaminase, glutamine phenylpyruvate transaminase), which presumably increased amino acid concentration in leaves and phloem sap under elevated CO₂. In contrast, aphid infested dnf1 plants had decreased activities of N assimilation‐related enzymes and transmination‐related enzymes and amino acid concentrations under elevated CO₂. Furthermore, elevated CO₂ up‐regulated expression of genes relevant to amino acid metabolism in bacteriocytes of aphids associated with Jemalong, but down‐regulated those associated with dnf1. Our results suggest that pea aphids actively elicit host responses that promote amino acid metabolism in both the host plant and in its bacteriocytes to favor the population growth of the aphid under elevated CO₂.     

‘Myrosin cells’ are not a prerequisite for aphid feeding on oilseed rape (Brassica napus) but affect host plant preferences

The enzyme myrosinase (EC 3.2.3.1.147) is present in specialised myrosin cells and forms part of the glucosinolate–myrosinase system, also known as ‘the mustard oil bomb’, which has an important role in the defence system of cruciferous plants against insect pests. Transgenic Brassica napus MINELESS have been produced by transgenic ablation of myrosin cells. This prompted us to investigate the importance of myrosin cells in plant–aphid interactions. In order to study this, we challenged transgenic MINELESS and wild‐type cultivar Westar seedlings with the aphids Brevicoryne brassicae (a specialist) and Myzus persicae (a generalist). Our study included aphid free‐choice and aphid fecundity experiments. Data from these experiments showed that B. brassicae prefers wild‐type seedlings and M. persicae prefers MINELESS. B. brassicae and M. persicae showed significant variation in establishment on plants regardless of whether they were wild type or MINELESS and also differed significantly in affecting plant parts. Myrosinase activity in MINELESS control seedlings was 83.6% lower than the wild‐type control seedlings. Infestation with either of the two aphid species induced myrosinase levels in both wild‐type and MINELESS seedlings. Infestation with M. persicae reduced the concentration of most glucosinolates while B. brassicae had the opposite effect. B. brassicae enhanced the formation of glucosinolate hydrolysis products both in wild‐type and MINELESS seedlings. However, M. persicae decreased All ITC but increased 3,4ETBut NIT in wild‐type seedlings. Taken together, the investigation shows that the presence of myrosin cells affects the preference of generalist and specialist aphid species for Brassica napus plants.     

Induction of aphid resistance in tobacco by the cucumber mosaic virus CMV∆2b mutant is jasmonate-dependent

Cucumber mosaic virus (CMV) is vectored by aphids, including Myzus persicae. Tobacco (Nicotiana tabacum ‘Xanthi’) plants infected with a mutant of the Fny strain of CMV (Fny-CMVΔ2b, which cannot express the CMV 2b protein) exhibit strong resistance against M. persicae, which is manifested by decreased survival and reproduction of aphids confined on the plants. Previously, we found that the Fny-CMV 1a replication protein elicits aphid resistance in plants infected with Fny-CMVΔ2b, whereas in plants infected with wild-type Fny-CMV this is counteracted by the CMV 2b protein, a counterdefence protein that, among other things, inhibits jasmonic acid (JA)-dependent immune signalling. We noted that in nontransformed cv. Petit Havana SR1 tobacco plants aphid resistance was not induced by Fny-CMVΔ2b, suggesting that not all tobacco varieties possess the factor(s) with which the 1a protein interacts. To determine if 1a protein-induced aphid resistance is JA-dependent in Xanthi tobacco, transgenic plants were made that expressed an RNA silencing construct to diminish expression of the JA co-receptor CORONATINE-INSENSITIVE 1. Fny-CMVΔ2b did not induce resistance to M. persicae in these transgenic plants. Thus, aphid resistance induction by the 1a protein requires JA-dependent defensive signalling, which is countered by the CMV 2b protein.     

Comparison of the potential rate of population increase of brown and green color morphs of Sitobion avenae (Homoptera: Aphididae) on barley infected and uninfected with Barley yellow dwarf virus

Life tables of brown and green color morphs of the English grain aphid, Sitobion avenae (Fabricius) reared on barley under laboratory conditions at 20 ± 1°C, 65% ± 5% relative humidity and a photoperiod of 16 : 8 h (L : D) were compared. The plants were either: (i) infected with the Barley yellow dwarf virus (BYDV); (ii) not infected with virus but previously infested with aphids; or (iii) healthy barley plants, which were not previously infested with aphids. Generally, both color morphs of S. avenae performed significantly better when fed on BYDV‐infected plants than on plants that were virus free but had either not been or had been previously infested with aphids. Furthermore, when fed on BYDV‐infected plants, green S. avenae developed significantly faster and had a significantly shorter reproductive period than the brown color morph. There were no significant differences in this respect between the two color morphs of S. avenae when they were reared on virus‐free plants that either had been or not been previously infested with aphids. These results indicate that barley infected with BYDV is a more favorable host plant than uninfected barley for both the color morphs of S. avenae tested, particularly the green color morph.     

The NIa‐Pro protein of Turnip mosaic virus improves growth and reproduction of the aphid vector,Myzus persicae (green peach aphid)

Many plant viruses depend on aphids and other phloem‐feeding insects for transmission within and among host plants. Thus, viruses may promote their own transmission by manipulating plant physiology to attract aphids and increase aphid reproduction. Consistent with this hypothesis, Myzus persicae (green peach aphids) prefer to settle on Nicotiana benthamiana infected with Turnip mosaic virus (TuMV) and fecundity on virus‐infected N. benthamiana and Arabidopsis thaliana (Arabidopsis) is higher than on uninfected controls. TuMV infection suppresses callose deposition, an important plant defense, and increases the amount of free amino acids, the major source of nitrogen for aphids. To investigate the underlying molecular mechanisms of this phenomenon, 10 TuMV genes were over‐expressed in plants to determine their effects on aphid reproduction. Production of a single TuMV protein, nuclear inclusion a‐protease domain (NIa‐Pro), increased M. persicae reproduction on both N. benthamiana and Arabidopsis. Similar to the effects that are observed during TuMV infection, NIa‐Pro expression alone increased aphid arrestment, suppressed callose deposition and increased the abundance of free amino acids. Together, these results suggest a function for the TuMV NIa‐Pro protein in manipulating the physiology of host plants. By attracting aphid vectors and promoting their reproduction, TuMV may influence plant–aphid interactions to promote its own transmission.     

Silencing of a lipase maturation factor 2‐like gene by wheat‐mediated RNAi reduces the survivability and reproductive capacity of the grain aphid,Sitobion avenae

Lipase maturation factor (LMF) family proteins are required for the maturation and transport of active lipoprotein lipases. However, the specific roles of LMF2 remain unknown. In this study, a grain aphid lmf2‐like gene fragment was cloned and was highly similar in sequence to a homologous gene in the pea aphid, Acyrthosiphon pisum. An RNAi vector was constructed with this fragment and used for wheat transformation. The expression of the lmf2‐like gene in aphid, as well as the growth and reproduction of the aphids, was analyzed after feeding on the transgenic wheat. There were no significant differences in the expression of the lmf2‐like gene over development. The expression of the lmf2‐like gene was significantly reduced by 27.6% on the fifth day, and 57.6% on the 10th day after feeding. The total number of aphids produced on the transgenic plants was less than the number produced on control plants, and the difference became significant or after 2 weeks. The molting numbers were also reduced in the aphids reared on the transgenic plants. Our findings indicate that lmf2‐like genes may have potential as a target gene for the control of grain aphids and show that feeding aphids with wheat expressing lmf2‐like RNAi resulted in significant reductions in survival and reproduction.     

Nitrogen fertiliser affects the functional response and prey consumption of Harmonia axyridis (Coleoptera: Coccinellidae) feeding on cereal aphids

Predator–prey interactions are influenced by nitrogen availability. Wheat (Triticum aestivum cv. Solstice) plants were provided with four levels of nitrogen and examined the responses of coccinellid predator, Harmonia axyridis to cereal aphids, Rhopalosiphum padi and Sitobion avenae. Increased nitrogen application improved nitrogen contents of the plants and also the body weight of cereal aphids feeding on them. In no‐choice feeding trials, H. axyridis consumed more aphids on low fertilised plants, suggesting a compensatory consumption to overcome reduced biomass (lower aphid size). Total biomass devoured by H. axyridis on all nitrogen fertiliser treatments was not statistically different. Logistic regression analysis of the proportion of prey consumed demonstrated that all developmental stages (larval and adult) of H. axyridis exhibited the type II functional response on all nitrogen fertiliser treatments. The rate of successful search (a′) of third and fourth instars and adults were the same across all fertiliser treatments suggesting that nitrogen fertilisation did not affect a′. Maximum handling time for the first instars of H. axyridis on R. padi (3.81 h^?1) and S. avenae (4.59 h^?1) was on the highest nitrogen treatment while minimum handling time was for the adults of H. axyridis on R. padi (0.20 h^?1) and S. avenae (0.20 h^?1) on the lowest nitrogen treatment. Handling time varied at varying fertiliser treatments within all instars and affected the predator's efficiency. The functional response curve, rate of successful search and handling time provide the information needed to understand the predator–prey interaction between H. axyridis and these cereals aphids. This could lead to the development of a better strategy for the biological control of R. padi and S. avenae at any particular level of nitrogen fertiliser regime in the field crops.     

Presence of Cry1Ab in the Bt maize – aphid (Rhopalosiphum maidis) – ladybeetle (Propylea japonica) system has no adverse effects on insect biological parameters

Transgenic Bacillus thuringiensis Berliner (Bt) crops receive particular attention because they carry genes encoding insecticidal proteins that might negatively affect non‐target arthropods. Here, laboratory experiments were conducted to evaluate the impact of Cry1Ab‐expressing transgenic maize [5422Bt1 (event Bt11) and 5422CBCL (MON810)] on the biological parameters of two non‐target arthropods, the aphid Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae) and its predator the ladybeetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae). In a long‐term assay (three generations), no significant differences were found between R. maidis fed Bt maize and those fed a near‐isogenic line (5422) when individual parameters were compared, including nymph development time, adult longevity, aphid spawning period, and fecundity. No negative effects were detected throughout the life cycle of P. japonica in aphids’ feeding amount, development (nymphs, pupae, adults, and progeny eggs), fecundity, or egg hatching when they preyed on Bt maize‐fed aphids compared with non‐Bt maize treatments. A tritrophic assay revealed that Cry1Ab was highly diluted through the food chain (Bt maize leaves, R. maidis, and P. japonica), as detected by an enzyme‐linked immunosorbent assay (ELISA). In conclusion, although Cry1Ab concentrations in maize leaves increased as the plants developed, Cry1Ab levels were significantly reduced in the aphid R. maidis, and no traces of Cry1Ab were detected in P. japonica preying on Bt maize‐fed aphids. The two hybrids of Bt maize expressing Cry1Ab had no negative effects on the measured biological parameters of the aphid R. maidis or its predator, the ladybeetle P. japonica.     

Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing

Dicer proteins are central to the different mechanisms involving RNA interference. Plants have evolved multiple DICER‐LIKE (DCL) copies, thus enabling functional diversification. In Arabidopsis, DCL2 and DCL4 process double‐stranded RNA into 22 and 21 nucleotide small interfering (si)RNAs, respectively, and have overlapping functions with regards to virus and transgene silencing. Nonetheless, some studies have reported that dcl2 or dcl4 single mutations are sometimes sufficient to hinder silencing. To better dissect the role of DCL2 and DCL4, we analyzed silencing kinetics and efficiencies using different transgenic systems in single and double mutant backgrounds. The results indicate that DCL2 stimulates transitivity and secondary siRNA production through DCL4 while being sufficient for silencing on its own. Notably, silencing of 35S‐driven transgenes functions more efficiently in dcl4 mutants, indicating that DCL4 mostly obscures DCL2 in wild‐type plants. Nonetheless, in a dcl4 mutant compromised in phloem‐originating silencing, ectopically expressed DCL2 allows restoration of silencing, suggesting that DCL2 is not, or poorly, expressed in phloem. Remarkably, this ectopic DCL2 contribution to phloem‐originating silencing is dependent on the activity of RNA‐DEPENDENT RNA POLYMERASE6. These results indicate that, despite differences in the silencing activity of their small RNA products, DCL2 and DCL4 mostly act redundantly yet hierarchically when present simultaneously.     

Effect of plant nutrition on aphid size,prey consumption,and life history characteristics of green lacewing

Plant quality can directly and indirectly affect the third trophic level. The predation by all the instars of green lacewing, Chrysoperla carnea （S.） （Neuroptera： Chrysopidae） on the cereal aphids, Rhopalosiphum padi （L.）, and Sitobion avenae （F.） at varying nitrogen fertilizer levels was calculated under laboratory conditions. Wheat plants were grown on four nitrogen fertilizer levels and aphids were fed on these plants and subsequently offered as food to the C. carnea. Aphid densities of 10, 30, and 90 were offered to first, second, and third instar larvae of green lacewing. Increased nitrogen application improved nitrogen contents of the plants and also the body weight of cereal aphids feeding on them. Aphid consumption by green lacewings was reduced with the increase in nitrogen content in the host plants of aphids. Predation of both aphid species by first, second, and third instars larvae of C. carnea was highest on aphids reared on plants with the lowest rate of fertilization, suggesting a compensatory consumption to overcome reduced biomass （lower aphid size）. Total biomass devoured by C. carnea on all nitrogen fertilizer treatments was not statistically different. Additionally, the heavier host prey influenced by the plant nutrition had an effect on the life history characteristics of green lacewings. The larval duration, pupal weight, pupal duration, fecundity, and male and female longevity were significantly affected by the level of nitrogen fertilization to the aphid＇s host plants, except for pupal duration when fed on S. avenae. This study showed that quantity of prey supplied to the larvae affects the prey consumption and thereafter the life history characteristics of green lacewings.     

Performance of clones and morphs of two cereal aphids on wheat plants with high and low nitrogen content

The great variability of the aphid life cycle and their tendency for host alternation gives rise to aphid clones and morphs. Inter‐ and intraclonal variability may be observed in the responses of aphids to various environmental factors. In this study we aimed to evaluate the influence of intrinsic factors (clone and morph type) on the intrinsic rate of increase (r_m) of the English grain aphid, Sitobion avenae (Fabricius), and the bird cherry‐oat aphid, Rhopalosiphum padi (Linnaeus). For each species four apterous clones were collected from established laboratory colonies and compared to assess their relative fitness on high‐ and low‐nitrogen wheat plants under laboratory conditions. The clones had significantly different intrinsic rates of increase on high‐ and low‐nitrogen plants. All R. padi clones had a higher intrinsic rate of increase and mean relative growth rate than S. avenae. Experiments were also conducted to compare the mean fecundity of apterous and alate morphs of S. avenae and R. padi clones on high‐ and low‐nitrogen wheat plants. On high‐nitrogen plants the apterous morphs of S. avenae clones had significantly higher mean fecundity than alate morphs. There were no significant differences between the mean fecundity of alate morphs of the same species on high‐ and low‐nitrogen plants. The results support the idea of better fitness of specific clones/morphs on certain host plants due to higher and lower intrinsic rates of increase.