A novel nematode effector suppresses plant immunity by activating host reactive oxygen species‐scavenging system

Summary Evidence is emerging that plant‐parasitic nematodes can secrete effectors to interfere with the host immune response, but it remains unknown how these effectors can conquer host immune responses. Here, we depict a novel effector, MjTTL5, that could suppress plant immune response. Immunolocalization and transcriptional analyses showed that MjTTL5 is expressed specifically within the subventral gland of Meloidogyne javanica and up‐regulated in the early parasitic stage of the nematode. Transgenic Arabidopsis lines expressing MjTTL5 were significantly more susceptible to M. javanica infection than wild‐type plants, and vice versa, in planta silencing of MjTTL5 substantially increased plant resistance to M. javanica. Yeast two‐hybrid, coimmunoprecipitation and bimolecular fluorescent complementation assays showed that MjTTL5 interacts specifically with Arabidopsis ferredoxin : thioredoxin reductase catalytic subunit (AtFTRc), a key component of host antioxidant system. The expression of AtFTRc is induced by the infection of M. javanica. Interaction between AtFTRc and MjTTL could drastically increase host reactive oxygen species‐scavenging activity, and result in suppression of plant basal defenses and attenuation of host resistance to the nematode infection. Our results demonstrate that the host ferredoxin : thioredoxin system can be exploited cunningly by M. javanica, revealing a novel mechanism utilized by plant–parasitic nematodes to subjugate plant innate immunity and thereby promoting parasitism.

Methods S1 Protoplast isolation and transformation.

Methods S2
The generation of constructs used in Protoplast transformation.

Methods S3
The generation of transgenic tomato roots expressing AtNTRc-mCherry.  Danio rerio Q06S87 *Named as AtTTL by Nam & Li (2004). However, the amino acid sequence analysis showed that the protein contain a 5-hydroxyisourate hydrolase domain, not contain DUF290 domains. Therefore, it should be transthyretin-related protein (TRP), namely AtTRP. In addition, CbTTL, CeTTL and BmTTL were listed as CBR-TTR-41, TTR-41 and BM-TTR-41 in the NCBI database, but the amino acid sequence analysis showed that these proteins contain a DUF290 domain, not contain a 5-hydroxyisourate hydrolase domain. Therefore we renamed them as CbTTL, CeTTL and BmTTL, respectively. The clone 1 and clone 5 contain the sequence from 1 st to 149 th and from 253 rd to 509 th of the NP_199834; the clone 2 and clone 4 contain the sequence from 1 st to 210 th and from 1 st to 87 th of the NP_001119099. The clone 2, 4, 5, 6, 7, 8 were found once, the clone 3 was found twice with identical sequence and the clone 1 was found three times with identical sequence.

Fig. S13 Subcellular localization of AtFTRc and MjTTL5. AtFTRc and MjTTL5
cDNA were fused in frame with the coding sequence of green fluorescent protein (GFP), respectively, and expressed in tomato root protoplast. The AtNTRc is a plastid-localized protein and used as a marker to indicated the plastid. Free GFP was used as control, CK represents the protoplast was not transformed any construct. Bar, 50 μm.

Fig. S14 Scrutinizing the interaction between AtFTRc homologs and MjTTL5 homologs. (a) Scrutinizing the interaction between different TTL homologs from
Meloidogyne spp. and AtFTRc; (b) scrutinizing the interaction between different TTL homologs from Radopholus similis and AtFTRc; (c) scrutinizing the interaction between MjTTL5 and AtFTRv or CeTTL and AtFTRc. The coding sequence of MjTTL1-MjTTL5, RsTTL1-RsTTL5, MiTTL5 and MeTTL5 were separately cloned into the bait vector pGBK and co-transformed with the prey vector pGAD contained AtFTRc, and the bait vector pGBK containing MjTTL5 was co-transformed with the prey vector pGAD containing AtFTRv into the yeast strain AH109. pGBK containing LamC was co-transformed with pGAD containing AtFTRc as a negative control.

Fig. S15 Expression pattern of AtFTRc transcripts in multiple plant tissues. Total
RNAs were extracted from leaves, flowers and roots from 30-day old wild type Arabidopsis. The AtActin gene (AT1G49240) was used as an internal control and the relative fold change was relative to the expression of roots. Error bars, ± SD.
5. The expression of AtNTRc-mCherry in tomato roots was confirmed by fluorescence microscope observation.