Sieve element occlusion provides resistance against Aphis gossypii in TGR-1551 melons.

Feeding behavior and plant response to feeding were studied for the aphid Aphis gossypii Glover on susceptible and resistant melons (cv. Iroquois and TGR-1551, respectively). Average phloem phase bout duration on TGR-1551 was <7% of the duration on Iroquois. Sixty-seven percent of aphids on TGR-1551 never produced a phloem phase that attained ingestion (EPG waveform E2) in contrast to only 7% of aphids on Iroquois. Average bout duration of waveform E2 (scored as zero if phloem phase did not attain E2) on TGR-1551 was <3% of the duration on Iroquois. Conversely, average bout duration of EPG waveform E1 (sieve element salivation) was 2.8 times greater on TGR-1551 than on Iroquois. In a second experiment, liquid nitrogen was used to rapidly cryofix leaves and aphids within a few minutes after the aphids penetrated a sieve element. Phloem near the penetration site was then examined by confocal laser scanning microscopy. Ninety-six percent of penetrated sieve elements were occluded by protein in TGR-1551 in contrast to only 28% in Iroquois. Usually in TGR-1551, occlusion was also observed in nearby nonpenetrated sieve elements. Next, a calcium channel blocker, trivalent lanthanum, was used to prevent phloem occlusion in TGR-1551, and A. gossypii feeding behavior and the plant's phloem response were compared between lanthanum-treated and control TGR-1551. Lanthanum treatment eliminated the sieve element protein occlusion response and the aphids readily ingested phloem sap from treated plants. This study provides strong evidence that phloem occlusion is a mechanism for resistance against A. gossypii in TGR-1551.

Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance.

We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.

The Conformation of a Plasma Membrane-Localized Somatic Embryogenesis Receptor Kinase Complex Is Altered by a Potato Aphid-Derived Effector.

Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding.

A novel virus from Macrosiphum euphorbiae with similarities to members of the family Flaviviridae.

A virus with a large genome was identified in the transcriptome of the potato aphid (Macrosiphum euphorbiae) and was named Macrosiphum euphorbiae virus 1 (MeV-1). The MeV-1 genome is 22 780 nt in size, including 3' and 5' non-coding regions, with a single large ORF encoding a putative polyprotein of 7333 aa. The C-terminal region of the predicted MeV-1 polyprotein contained sequences with similarities to helicase, methyltransferase and RNA-dependent RNA polymerase (RdRp) motifs, while the N-terminal region lacked any motifs including structural proteins. Phylogenetic analysis of the helicase placed MeV-1 close to pestiviruses, while the RdRp region placed it close to pestiviruses and flaviviruses, suggesting MeV-1 has a positive-polarity ssRNA genome and is a member of the family Flaviviridae. Since the MeV-1 genome is predicted to contain a methyltransferase, a gene present typically in flaviviruses but not pestiviruses, MeV-1 is likely a member of the genus Flavivirus. MeV-1 was present in nymphal and adult stages of the aphid, aphid saliva and plant tissues fed upon by aphids. However, the virus was unable to multiply and spread in tomato plants. In addition, dsRNA, the replication intermediate of RNA viruses, was isolated from virus-infected M. euphorbiae and not from tomato plants infested with the aphid. Furthermore, nymphs laid without exposure to infected plants harboured the virus, indicating that MeV-1 is an aphid-infecting virus likely transmitted transovarially. The virus was present in M. euphorbiae populations from Europe but not from North America and was absent in all other aphid species tested.

The tomato leucine-rich repeat receptor-like kinases SlSERK3A and SlSERK3B have overlapping functions in bacterial and nematode innate immunity.

The Somatic Embryogenesis Receptor Kinase 3 (SERK3)/Brassinosteroid (BR) Insensitive 1-Associated Kinase 1 (BAK1) is required for pattern-triggered immunity (PTI) in Arabidopsis thaliana and Nicotiana benthamiana. Tomato (Solanum lycopersicum) has three SlSERK members. Two of them exhibit particularly high levels of sequence similarity to AtSERK3 and, therefore, were named SlSERK3A and SlSERK3B. To characterize a role for SlSERK3A and SlSERK3B in defense, we suppressed each gene individually or co-silenced both using virus-induced gene silencing (VIGS) in the tomato cv. Moneymaker. Co-silencing SlSERK3A and SlSERK3B resulted in spontaneous necrotic lesions and reduced sensitivity to exogenous BR treatment. Silencing either SlSERK3A or SlSERK3B resulted in enhanced susceptibility to root knot-nematode and to non-pathogenic Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC indicating that both SlSERK3s are positive regulators of defense. Interestingly, silencing SlSERK3B, but not SlSERK3A, resulted in enhanced susceptibility to the pathogenic strain Pst DC3000 indicating distinct roles for these two SlSERK3 paralogs. SlSERK3A and SlSERK3B are active kinases, localized to the plasma membrane, and interact in vivo with the Flagellin Sensing 2 receptor in a flg22-dependent manner. Complementation of the Atserk3/bak1-4 mutant with either SlSERK3A or SlSERK3B partially rescued the mutant phenotype. Thus, SlSERK3A and SlSERK3B are likely to constitute tomato orthologs of BAK1.

The receptor-like kinase SlSERK1 is required for Mi-1-mediated resistance to potato aphids in tomato.

The plant receptor-like kinase somatic embryogenesis receptor kinase 3 (SERK3)/brassinosteroid insensitive 1-associated kinase 1 (BAK1) is required for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Here we show that a distinct member of the SERK family, SERK1, is required for the full functioning of Mi-1, a nucleotide binding leucine-rich repeat (NB-LRR) resistance protein. Mi-1 confers resistance to Meloidogyne spp. (root-knot nematodes, RKNs) and three phloem-feeding insects, including Macrosiphum euphorbiae (potato aphid). SERK1 was identified in a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) screen in Nicotiana benthamiana. The screen was based on the suppression of a pest-independent hypersensitive response triggered by a constitutively active form of Mi-1, Mi-DS4. To assess the role of SERK1 in Mi-1-mediated resistance, Solanum lycopersicum (tomato) SlSERK genes were cloned. Three SlSERK members were identified with homologies to Arabidopsis AtSERK1 or AtSERK3/BAK1, and were named SlSERK1, SlSERK3A and SlSERK3B. SlSERK1 is ubiquitously expressed in tomato. Reducing SlSERK1 transcript levels in resistant plants, using gene-specific TRV-SERK1 VIGS, revealed a role for SlSERK1 in Mi-1-mediated resistance to potato aphids, but not to RKNs. In addition, Mi-1-dependent SlWRKY72 gene regulation was compromised in SlSERK1-silenced plants, placing SlSERK1 in the Mi-1 signaling pathway. Silencing SlSERK1 in a susceptible tomato background did not reduce the susceptibility to aphids, indicating that SlSERK1 is unlikely to be an essential virulence target. SlSERK1 is an active kinase, mainly localized at the plasma membrane. This work identifies a critical early component of Mi-1 signaling, and demonstrates a role for SlSERK1 in NB-LRR-mediated immunity.