Primary biological function of NLPs is unlikely related to the virulence of plant

Bailey (1996) identified the Nep1 protein from the plant-pathogenic fungus Fusarium oxysporum.

Therefore, the biological role of NLPs-family was originally considered to be related to fungal virulence on their host plants. However, their follow-up studies failed to confirm this hypothesis, because the disruption of NEP1 protein of F. oxysporum didn’t affect its virulence on cocoa (Bailey et al. 2002). Since then, the role of the NLP protein family turned mysterious.

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In the following two decades, many research groups tried to verify the relationship between NLPs and pathogenicity towards their host plants. However, the diverse results of these studies did not allow clear conclusions. Table 9 summarizes the most important studies on effects that are caused by NLPs gene-disruption on pathogen virulence. It indicates that some members of NLP-family contribute to virulence on specific host plants, while some do not.

Firstly, the deletion of NLPs from some pathogens compromised their virulence, which was confirmed for fungi, bacteria and oomycetes. Disruption of the EccNip from the soft-rot bacteria Erwinia carotovora subsp. carotovora reduced its virulence on potato tubers, but not on potato stem (Mattinen et al. 2004). Three NLP-genes in Phytophthora capsici were shown to be required for full virulence of oomycetes (Feng et al. 2014). Reduction of Ssnep2 gene expression from Sclerotinia sclerotiorum resulted in decreased virulence on its host plant, Brassica napus (Dallal Bashi et al. 2010). Deletion of PeNLP1, but not PeNLP2, affected the virulence of post-harvest pathogen Penicillium expansum on apple fruits (Levin et al. 2019). Remarkably, disruption of either VdNLP1 (equal to VlNLP1) or VdNLP2 (equal to VlNLP6) genes encoding cytotoxic NLPs from Verticillium dahliae compromised its virulence on tomato and Arabidopsis, but only deletion of VdNLP1, not VdNLP2, affected its virulence on Nicotiana benthamiana (Santhanam et al. 2013). More interestingly, neither VdNLP1 nor VdNLP2 are essential to V. dahliae virulence on cotton plants. Even the simultaneous disruption of both genes failed to reduce virulence (Zhou et al. 2012). NLPs appear to have host-specific functions. It is noticeable, however, the reduced plant symptom representing less virulence may not always have biological meaningful in reality, for example, 15% less diameter of the lesion.

The virulence reduction was also observed in our previous study of VlNLP. Beinhoff (2011) showed that disruption of VlNLP1 resulted in a partial loss of virulence of VL43 on its host Brassica napus. B. napus plants infected by VlNLP1 knockout V. longisporum isolates were obviously higher than the plants infected by wild type fungi.

However, other studies provided opposite results. The wheat pathogen Mycosphaerella graminicola contains only a single NLP, but the NLP-deletion strain was equal to virulent (Motteram et al. 2009). Researches on two Botrytis fungi, Botrytis cinerea and Botrytis elliptica, showed that the disruption of NLPs genes failed to influence the virulence on their hosts tomato and lily (Cuesta Arenas et al. 2010; Staats et al. 2007). Even if the four MoNLPs in Magnaporthe oryzae had been disrupted together, virulence remained intact (Fang et al. 2017).

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Certain conditions are required for NLP genes expression in some microorganisms. For example, Nep gene from the bacteria Erwinia carotovora was only expressed after the contact with solid medium (Mattinen et al. 2004). In contrast, except VdNLP5, most VdNLPs of the cotton-pathogen V. dahliae (V592) were expressed in fungal hyphae or spores without requiring specific conditions (Zhou et al. 2012). Our results indicate that VlNLPs have a similar expression pattern as VdNLPs, which is that VlNLP3 (equal to VdNLP5) gene is also not expressed in the VL43 isolate when cultured in SXM.

Current researches studies mainly focus on the expressed NLPs, but ignore the fact that a variety of NLP genes are suppressed. For instance, 13 out of 33 NLP genes are suppressed in P. sojae (Dong et al. 2012), whereas 4 of 12 NLPs from H. arabidopsidis are also not expressed during infection (Cabral et al. 2012). More interestingly, our study shows that VlNLP3 suppression is strain-specific in VL43. Its expression was only detected in the isolates that belong to the A1/D1 and A1/D3 lineages including VL787, VL1194 and VL32. In addition to lineages difference, VlNLP3 expression is possibly related to fungal virulence as well. The preliminary results indicated that avirulent isolates may have better capability to express VlNLP3, which matches to our assumption that suppression of VlNLP3 expression is a prerequisite for pathogenicity.

Although VL1194 is an oilseed rape virulent strain, its VlNLP3 transcript level is much lower than in the avirulent VL32 isolate from the same A1/D3 lineage as VL1194.

Unexpectedly, in the root-dipping experiment, the oilseed rape plants inoculated with VlNLP3 overexpressed VL43 spore suspension failed to show any disease severity reduction compared with plants infected by the wild type strain, neither disease symptoms nor fungal DNA amounts in plant hypocotyl tissue. According to these results, VlNLP3 is not involved in pathogenicity.

In conclusion, the current insights of the role of NLP protein family are still controversial. The disruption of NLP genes failed to affect fungal virulence in many cases. Conclusively, the obtained data suggest that the original biological role of NLPs may not be related to plant pathogenicity. Besides, the study of VdNLPs from V. dahliae can also be involved in fungal vegetative growth and conidiospore production, but these results are only supported by a single published study (Santhanam et al. 2013).

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Table 9 Effect of the disruption of NLP genes on virulence of pathogens Organisms Disrupted gene Virulence effect

(Host plant specific)

Cotton¹ Tomato² Arabidopsis² N. benthamiana² Verticillium dahliae Sclerotinia sclerotiorum ∆SsNep1 No

∆SsNep2 Yes Wheat⁷ Mycosphaerella graminicola ∆MgNLP No

Apple fruit⁸

Penicillium expansum ∆PeNLP1 Yes

∆PeNLP2 No

Botrytis elliptica ∆BeNEP1 No

∆BeNEP2 No

References: 1: Dou and Zhou (2012); 2:Santhanam et al. (2013); 3.Mattinen et al. (2004); 4.Cuesta Arenas et al. (2010); 5.Bailey et al. (2002); 6.Dallal Bashi et al. (2010); 7.Motteram et al. (2009); 8.Levin et al. (2019); 9.Fang et al. (2017); 10.Feng et al.

(2014); 11. Staats et al. (2007)