Heterodera avenae, the most commonly reported species of cereal cyst nematode (CCN), causes substantial crop yield losses of 30–100% in wheat production worldwide (Bonfil et al., 2004; Nicol et al., 2007). The infective second-stage juveniles (J2s) of CCN invade the lateral roots or root tips of plants and migrate intracellularly toward the vascular cylinder, where they form feeding sites called syncytia. The CCN becomes sedentary and remains associated with the developing syncytium from which it obtains nutrients necessary for growth and development (Sobezak and Golinowski, 2009). A better understanding of the mechanisms underlying the interactions between CCN and its hosts is important for the development of new control strategies.

Plants respond to infection using two modes of innate immunity (Jones and Dangl, 2006; Tsuda and Katagiri, 2010). The first mode of immunity is referred to as “pattern-triggered immunity” (PTI) and is triggered by microbe-associated or pathogen-associated molecular patterns (MAMPs or PAMPs). Through evolution, adapted pathogens secrete effector proteins into plant cells and suppress PTI (Boller and He, 2009). To counter pathogens, plants have evolved resistance (R) proteins that specifically recognize certain pathogen effectors, resulting in initiation of the second mode of plant immunity, which is referred to as “effector-triggered immunity” (ETI) (Chisholm et al., 2006). The dynamic co-evolution of plants and pathogens is ongoing, and some pathogens have acquired effectors that interfere with ETI (Jones and Dangl, 2006). Thus, plants and microbial pathogens are engaged in an endless “arms race.”

Plant pathogens secrete proteins and other molecules known as effectors that modulate plant defenses and permit the pathogens to colonize plant tissue (Hogenhout et al., 2009). Systematic identification and characterization of the effectors that regulate plant immunity has been reported for various pathogens. Agro-infiltration in tobacco is the most popular assay used in the identification of genes regulating plant immunity because it is simple and quick. In bacteria, Pseudomonas syringae pv. tomato DC3000 expresses 36 bacterial type III effectors, 32 of 35 tested effectors can suppress HopA1-dependent ETI in tobacco, and many effectors can also suppress PTI (Guo et al., 2009). In oomycetes, the avirulence homolog (Avh) proteins share RXLR-dEER motifs, rendering them all candidate effectors. Of 169 effectors tested, most of the Avh proteins identified in the Phytophthora sojae genome suppress BAX-triggered programmed cell death (BT-PCD) using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana (Wang et al., 2011). In the fungus Ustilaginoidea virens, more than half of 30 randomly selected putative effectors identified in the genome were found to suppress the Burkholderia glumae-triggered hypersensitive reaction (HR) in N. benthamiana (Zhang et al., 2014). In addition to functioning as virulence factors that cripple host defenses, some effectors have been demonstrated to trigger host immunity. Eleven of 169 effectors in P. sojae were shown to trigger cell death, chlorosis, or mottling in N. benthamiana leaves (Wang et al., 2011). Among 42 Magnaporthe oryzae effectors identified in infected rice leaves, five proteins induced cell death in rice protoplasts only when they contained a signal peptide (SP) (Chen et al., 2013). Eight of 119 putative effectors from U. virens were proven to trigger cell death in rice protoplasts, and the SP of these proteins are essential for their cell-death-inducing activity (Fang et al., 2016). Recently, bioinformatic analyses of the draft genome sequences and transcriptome in plant parasitic nematodes have identified a lot of candidate effector proteins (Abad et al., 2008; Opperman et al., 2008; Thorpe et al., 2014; Eves-van den Akker et al., 2016; Li et al., 2016), which provide resources for large scale identification of nematodes effectors with the ability to suppress or induce plant defenses.

Unlike bacterial and oomycete effectors, a limited number of plant parasitic nematode (PPN) effectors have been functionally characterized. Some nematode effectors have been found to suppress host immunity through various molecular mechanisms (Haegeman et al., 2012; Favery et al., 2016). Overexpression of Meloidogyne incognita calreticulin in Arabidopsis thaliana suppresses the induction of defense marker genes and callose deposition after treatment of the plant with the PAMP elf18 (Jaouannet et al., 2013). Mj-FAR-1, a fatty acid- and retinol-binding protein secreted by M. javanica, has been shown to interfere with host lipid-based defenses and thereby facilitate infection (Iberkleid et al., 2013). MjTTL, a transthyretin-like homolog from M. javanica, exploits the host’s ferredoxin: thioredoxin system and drastically increases host reactive oxygen species-scavenging activity, resulting in suppression of plant basal defenses (Lin et al., 2016). The M. incognita putative secretory protein MiMsp40 suppresses programmed cell death (PCD) triggered by BAX, MAPK cascades and the ETI elicitors R3a/Avr3a, and overexpression of MiMsp40 in plants suppresses the deposition of callose and the expression of PTI marker genes (Niu et al., 2016). The CEP12 peptide from Globodera rostochiensis suppresses resistance-gene-mediated cell death, thereby suppressing plant immunity (Chronis et al., 2013). Several members of the SPRYSEC effector family in G. rostochiensis function as selective suppressors of defense-related PCD (Diaz-Granados et al., 2016). VAPs from G. rostochiensis and H. schachtii only affected the programmed cell death mediated by surface-localized immune receptors (Lozano-Torres et al., 2014). HgGLAND18 from H. glycines strongly suppresses both basal and hypersensitive cell death innate immune responses, and immunosuppression requires the presence and coordination between multiple protein domains (Noon et al., 2016). Annexin from H. avenae can suppress PCD triggered by BAX and the induction of marker genes of PTI in N. benthamiana (Chen et al., 2015). Moreover, effectors that induce plant defenses have also been described in nematodes. GrEXPB2 inhibits the cell death induced by PiNPP, AtRX and AvrBs2/Bs2 in tobacco leaves, and also induces chlorosis in N. benthamiana and cell death in tomato and potato. GrEXPB2 may have the dual properties of suppressing and eliciting plant defenses (Ali et al., 2015). Cg1 from M. javanica appears to be involved in triggering the immune response in host plants carrying the Mi-1 resistance gene (Gleason et al., 2008). The effector protein RBP-1 of G. pallida is reported to elicit cell death through the NB-LRR protein Gpa2 (Sacco et al., 2009). Transient expression of the G. rostochiensis effector VAP1 in tomato plants harboring Cf-2 and Rcr3^pim triggers a defense-related PCD in plant cells (Lozano-Torres et al., 2012). HaEXPB2, a predicted expansin-like protein from H. avenae, causes cell death when expressed with the SP in N. benthamiana (Liu et al., 2016). However, neither large-scale identification of nematode effectors that regulate plant immunity nor cooperation between effectors has been reported to date.

RNA silencing is a major type of defense mechanism against RNA viruses (Ding, 2010). Viral infection induces host siRNAs that direct the cleavage of viral RNAs. To counter this type of defense, viruses have developed suppressors of viral RNA silencing that interfere with the RNA silencing machinery and promote infection (Incarbone and Dunoyer, 2013). However, viruses are not the only organisms that are able to promote infection by blocking RNA silencing. Three known bacterial effectors can suppress the miRNA pathway in A. thaliana (Navarro et al., 2008). Two oomycete RxLR effectors, PSR1 and PSR2, of P. sojae were shown to suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs (Qiao et al., 2013). A PSR2-like effector from the related species P. infestans can also suppress RNA silencing (Xiong et al., 2014). Recently, it has also been reported that transgenic tobacco plants expressing a known viral suppressor display increased susceptibility to root-knot nematode (RKN) infection and that RKN parasitism may suppress host RNA silencing (Walsh et al., 2017). To date, however, no suppressors of RNA silencing have been reported in nematodes.

As a biotrophic pathogen, H. avenae needs to suppress plant defenses during the entire parasitic process. How many effectors of H. avenae are involved in suppressing plant immunity? What is the interplay between the effectors? Recently, the transcriptomes of H. avenae during infection of wheat and incompatible hosts have been published (Kumar et al., 2014; Zheng et al., 2015; Yang et al., 2017), thereby providing a resource for the large-scale mining of effectors in H. avenae. In this study, we systematically investigated the ability of a large number of putative effectors encoded in the H. avenae transcriptome to suppress plant defenses. The results revealed that most of the putative effectors had the potential to suppress PCD triggered by BAX, effectors or elicitin. Interestingly, several of the putative effectors could trigger cell death in N. benthamiana by themselves. We also explored the interplay between effectors that suppress PCD and those that induce PCD. Our results provide important information to understand how nematodes regulate plant defenses.

The effector proteins secreted by PPNs play essential roles in host–pathogen interactions (Mitchum et al., 2013). Transient expression assays in the N. benthamiana model system employing agroinfiltration have been successfully used to identify many bacterial, oomycete, fungal and nematode effectors (Guo et al., 2009; Wang et al., 2011; Zhang et al., 2014; Ali et al., 2015). In this study, we cloned 95 putative effectors from an effector repertoire identified by bioinformatics analysis of the H. avenae transcriptome. We found that 78 putative effectors suppressed PCD triggered by BAX and 7 putative effectors induced cell death or chlorosis in N. benthamiana (Figure 1A and Supplementary Table S2). The two-step Agro-infiltration protocol may result in that the prior infiltration inhibit subsequent T-DNA transfer, but the level of BAX protein was identical in tissues showing suppression and those not showing suppression (Wang et al., 2011). So the suppression of BT-PCD did not result from suppression of BAX DNA delivery or PVX massive replication after the second A. tumefaciens infiltration. In addition, although our assays were conducted in the non-host N. benthamiana, many studies of other pathogen effectors have shown that these effectors possess the same ability to suppress or induce cell death in non-host and host plants. For example, Avh172 and Avh6 of P. sojae were shown to suppress ETI in both the non-host N. benthamiana and in host soybean (Wang et al., 2011). Of the five M. oryzae cell-death-inducing proteins, all induce cell death in both host rice and non-host maize protoplasts, and four also cause cell death in the protoplasts of the dicot plants A. thaliana and N. benthamiana (Chen et al., 2013). These results demonstrate that the plant defenses machinery is well-conserved in diverse plant families. Accordingly, the transient expression assay in N. benthamiana is an efficient method for preliminary large-scale screening of putative effectors that may suppress or induce cell death. Further investigations (such as ROS assay, callose deposition, immunization-related gene expression. etc.) will be performed to verify the plant immunity suppressing abilities of the putative effectors.

To ascertain the characteristics such as the secretion ability and the role in parasitism of the selected candidate effectors, experimental confirmation was conducted. The yeast secretion assay has been used to investigate the secretion of predicted effectors (Chen et al., 2013; Fang et al., 2016). Using this assay, we verified that 33 of 53 predicted SPs of H. avenae putative effectors were functional in the yeast secretion system (Supplementary Table S2). Furthermore, the developmental expression pattern results demonstrated that most of the 30 tested effector candidates of H. avenae were transcriptionally expressed at higher levels during parasitic stages than during pre-parasitic stages (Figure 2), indicating that these putative effectors play important roles in parasitism. In addition, the ten tested effectors were all observed in gland cells of H. avenae by in situ hybridization (Figure 3); thus, they display a major feature of nematode effector proteins. Previous research has shown that systemic expression of effectors in planta is an effective method that can be used to identify effectors that cause dramatic phenotypes in plants (Ali et al., 2015). In our work, 56 of 67 tested effector candidates induced symptoms during systemic expression in N. benthamiana; these symptoms included necrosis, wilting, dwarfing and aggravation of PVX symptoms (Figure 5 and Supplementary Table S2), demonstrating that the putative effectors may play some roles in plants.

Recently, several suppressors of RNA silencing, a common counter-defense strategy used by viruses, have been reported among bacterial and oomycete pathogens (Navarro et al., 2008; Qiao et al., 2013). Suppression of the RNA silencing pathway is observed during RKN infection of N. tabacum, but no specific suppressors of RNA silencing have been identified (Walsh et al., 2017). We attempted to find suppressors of RNA silencing in H. avenae by agroinfiltration of N. benthamiana. However, when the 52 tested candidate effectors of H. avenae were co-infiltrated with eGFP, we observed no suppression of RNA silencing (Supplementary Table S2). It would probably be more useful to screen more effector candidates for silencing suppressor activities.

PTI and ETI comprise two branches of plant defenses. Ten BT-PCD-suppressing effector candidates in H. avenae were randomly selected, and their ability to suppress PTI or ETI was tested. As expected, all of the selected effector candidates could also suppress PCD triggered by the elicitor PsojNIP (Figure 7B) and at least one R-protein/cognate effector pair (Figures 7D,F). PsojNIP acts as an elicitor in a manner similar to PAMPs, which induce necrosis at the extracytoplasmic side of the host plasma membrane (Qutob et al., 2006). The resistance proteins R3a and Gpa2 recognize their respective elicitors, Avr3a and RBP-1, intracellularly to induce ETI (Huang et al., 2005; Sacco et al., 2009). Our results indicated that all the tested putative effectors of H. avenae could suppress both PTI and ETI (Table 1), and they were active in suppressing cell death not only in apoplasts but also in the cytoplasm. The high proportion of putative effectors that were found to suppress PCD suggests that suppression of plant immunity is one of the primary ways in which H. avenae effectors contribute to biotrophic parasitism. Our results are consistent with previous findings in P. syringae and P. sojae that most tested effectors can suppress plant defenses (Guo et al., 2009; Wang et al., 2011).

Biotrophic pathogens usually avoid triggering plant cell death; instead, they typically suppress plant defenses for survival. Surprisingly, 7 of 95 putative effectors in H. avenae were shown to have the ability to induce cell death in N. benthamiana (Figure 1B), indicating that these proteins might be recognized by the plant defenses machinery or might function as toxins to induce necrosis. For hemibiotrophic plant pathogens, which undergo a necrotrophic phase following the biotrophic phase, some effectors in P. sojae and M. oryzae have been shown to induce cell death (Wang et al., 2011; Chen et al., 2013). These cell-death-inducing effectors may facilitate the colonization of pathogens during the late necrotrophic phase of infection. To date, in nematodes, only expansin and expansin-like proteins (GrEXPB2, HaEXPB1 and HaEXPB2) have been reported to act alone to trigger cell death in plants (Ali et al., 2015; Liu et al., 2016). These proteins fail to induce cell death when their SPs are deleted, indicating that they only function when present in the apoplastic space of the plant cell. Expansins located outside the cell may bind to and alter the plant cell wall, resulting in cell death (Liu et al., 2016). In contrast, in our study, the 7 cell-death-inducing putative effectors all functioned in the absence of SPs. Therefore, these putative effectors appear to trigger cell death by acting within the cytoplasm of the plant cell. However, the functions of the cell-death-inducing effectors in biotrophic H. avenae remain to be elucidated.

Interestingly, cooperation between effectors that suppress PCD and those that induce PCD was observed in H. avenae. Of the ten putative effectors that suppressed BT-PCD, nine also suppressed PCD triggered by the cell-death-inducing putative effectors themselves (Table 2). This result is consistent with the hypothesis that biotrophic nematodes must avoid inducing host defenses for successful parasitism. In G. pallida, the effector RBP-1 is recognized by the potato resistance protein Gpa2 and elicits HR, whereas two SPRYSECs of G. pallida can suppress the induced cell death (Mei et al., 2015). Cooperation between effectors was also reported in P. sojae, in which it was shown that all of the effectors that suppress BT-PCD could also suppress PCD triggered by at least one effector (Avh238 and Avh241) (Wang et al., 2011). Although the induction of cell death is related to the triggering of plant defenses mechanisms, it has been reported that cell death induction might not contribute to host resistance. HaEXPB2 induces cell death, but treatment with RNAi for HaEXPB2 does not significantly change the number of cysts produced in the host (Liu et al., 2016), suggesting that HaEXPB2 has no effect on host resistance. Similarly, we presumed that some cell-death-inducing effectors of H. avenae might be recognized by plants and induce defense responses, whereas other effectors cooperate in suppressing the induced defense responses. Through this cooperation, H. avenae ensures that the recognition of cell-death-inducing effectors does not confer resistance in plants.

Conserved domains are reported to be essential for the function of cell-death-inducing effectors (Chen et al., 2013; Fang et al., 2016). The entire HaEXPB2 protein, including the C-terminus, the β-expansin domain, and the carbohydrate-binding domain, is necessary for inducing cell death in N. benthamiana (Liu et al., 2016). Through a Pfam search, isotig12969 and isotig19390 were each predicted to contain a PROF (profilin) domain, which is typically involved in actin binding. Actin reorganization is crucial for the development and expansion of nematode feeding sites. When Mi131, a protein from M. incognita that bears a profilin domain, was expressed in protoplasts in which the actin cytoskeleton had been labeled with GFP, the actin cytoskeleton appeared fragmented (Leelarasamee, 2015). Profilin from the protozoan parasite Toxoplasma gondii plays a role in motility and serves as a microbial ligand that is recognized by the host innate immune system, functioning like bacterial flagellin (Plattner et al., 2008). In our study, isotig12969 and isotig19390 lacking the PROF domain lost the ability to induce cell death (Figure 8); this result is consistent with the above reports that profilin resembles PAMPs. Isotig16511 was predicted to be an endonuclease G, a type of endonuclease that acts as a PCD DNase when released from mitochondria (Li et al., 2001). As expected, isotig16511 lacking a NUC domain was unable to trigger cell death (Figure 8), consistent with the predicted function of this protein.

Although the tested H. avenae putative effectors in this study has been nearly one hundreds, it will be necessary to examine more effectors in the future. Our study provides a method for the large-scale and rapid investigation of the effector repertoire of PPN in the suppression of plant defenses mechanisms. Our data show that the majority of the effectors predicted based on transcriptomic analysis have the potential to suppress plant defenses, including PTI and ETI. This study reveals the important role of H. avenae effectors in biotrophic infection and significantly advances our understanding of the defense-suppressing function of PPN effectors.

HJ and QL conceived the idea, acquired funding and designed the experiments. CC, YC, LP, YD, FS, and SY performed the experiments. CC and DY contributed toward the data analyses. CC, QL, and HJ wrote and revised the manuscript. All authors read and approved the final version of the manuscript for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.