The present study suggests that small ncRNAs seem to be correlated with some effector proteins expressed in P. infestans during the hemibiotrophic life cycle as well as with other molecular regulatory processes. Most likely, small ncRNAs can be involved in one of the regulatory mechanisms mediating the infection processes of P. infestans in S. tuberosum. Moreover, a useful bioinformatic pipeline was presented and implemented for the analysis of small RNA-seq data sets in P. infestans.
One of the most relevant trends observed in the results obtained bioinformatically was that the small ncRNAs were associated with effector-like proteins. To start, crinkler and crinkler-like proteins, which are well-known effectors associated with the necrotrophic phase in P. infestans, were found to be downregulated at the later stages of the infection cycle (48 hpi and 72 hpi) when compared to in vitro mycelia. Therefore, if these “silencing” small ncRNAs are not expressed at later stages of the cycle, the expression of their corresponding genes will not be inhibited, and thus, the production of the associated proteins will be enabled. Since crinkler proteins are mostly associated with the necrotrophic phenotype observed in hemibiotrophic pathogens, this scenario is expected and provides evidence of the association of small ncRNAs of P. infestans with the regulation of the transition from biotrophic to necrotrophic stages [3, 31].
The RXLR effectors are almost always related to the biotrophic stage of the infection . Therefore, the finding that a small ncRNA associated with the Avr2 family secreted RxLR effector was significantly upregulated at the necrotrophic stage was expected. The expression of the gene associated with this small ncRNA was validated using qRT-PCR. In this experiment, it was evident that this gene was only slightly upregulated during biotrophy and then became downregulated during both the transition from biotrophy to necrotrophy and the necrotrophic phases. This finding supports the idea that the small ncRNA associated with the gene may be one of the regulatory mechanisms affecting the expression of this gene, especially during necrotrophy. Furthermore, it supports the idea that RXLR effectors are mostly expressed during the biotrophic stage, as suggested by Zuluaga and collaborators (2016).
Similarly, elicitins and elicitin-like proteins are relevant effectors expressed at different time points of the infection cycle. Some are known to be highly expressed during the biotrophic phase (early in the infection), while others are highly expressed during the necrotrophic phase (at later time points in the infection cycle) . In this study, we identified small ncRNAs associated with the elicitin-like proteins INF–1, INF–6, and INF–2A, which have been defined as important necrotrophic elicitors [3, 32] or as inducers of various degrees of the hypersensitive response or programmed cell death in plants . Small ncRNAs associated with these elicitin-like proteins were significantly downregulated at every time point when compared to mycelia grown in media. Thus, the genes associated with these small ncRNAs are expected to be overexpressed throughout the disease cycle. However, the genes associated with INF1 and INF6 were evaluated by qRT-PCR and were found to be downregulated during all stages of the cycle when compared to the in vitro mycelia, which does not support the expected expression profile of the genes. This highlights the complexity of the disease progression gene regulation.
A wide variety of small ncRNAs associated with enzymatic processes were found to be differentially expressed across the majority of the comparisons made. Their up- and downregulation is highly likely to be involved in how the pathogen responds to host defenses. One example is the sporangia-induced phosphatidylinositol kinase, which, as its name suggests, is usually induced in response to sporangia production in P. infestans. In this case, the small ncRNA associated with the gene seems to be upregulated at the beginning of the infection when compared to the transition from the biotrophy to the necrotrophy stage and to the in vitro mycelia (Table 1). Therefore, the associated gene would be expected to be downregulated during the biotrophic stage when compared to necrotrophy and to the in vitro mycelia. This is consistent with the function of this gene because P. infestans mainly feeds on the host during the biotrophic phase . Therefore, the production of sporangia is most likely to occur near the end of the necrotrophic stage when the pathogen needs to proliferate and disperse to other plants. The qRT-PCR analyses showed that this gene is downregulated in all stages when compared to the in vitro mycelia (this was less evident in biotrophy and necrotrophy than in the transition between them). This suggests that the isolate had not sporulated in the specific moment of the necrotrophic stage that was tested and potentially needed more time to sporulate.
In a similar sense, we evaluated the peptidyl-prolyl cis-trans isomerase, an enzyme that accelerates protein folding and may be part of a downstream reaction in the infection process. Based on the differential expression of its associated small ncRNAs (Table 1), the peptidyl gene would be expected to be overexpressed during biotrophy. This is because its small ncRNA was downregulated when comparing 24 hpi (biotrophy) to 48 hpi (transition to necrotrophy). The qRT-PCR results support an overexpression of the peptidyl-prolyl cis-trans isomerase gene during biotrophy followed by a downregulation during the transition to necrotrophy when compared to the in vitro mycelia.
The nuclear LIM factor interactor-interacting protein spore-specific form gene was predicted to be downregulated during biotrophy when compared to the in vitro mycelia because the corresponding small ncRNA was upregulated when these two conditions were compared (see Table 1). However, this gene was found to be ubiquitously upregulated in all stages of the infection when compared to the in vitro mycelia based on the qRT-PCR assays. In theory, these types of proteins are zoosporogenesis induced, and they seem to perform a role of phosphatases when P. infestans is producing zoospores [34, 35]. Thus, they are expected to be upregulated at later stages of the infection when the zoospores are being produced. However, the pattern obtained does not clearly support the involvement of small ncRNAs in the regulation of the expression of this gene.
Berberine-like proteins are known to be involved in alkaloid biosynthesis and hydrogen peroxide production . Hence, they are important virulence factors during plant infection and could also perform a protective role in Phytophthora against the plant defenses . A Berberine-like protein gene would be expected to be downregulated when comparing its expression during necrotrophy with that of in vitro mycelia. This is because its corresponding small ncRNA was upregulated when comparing these two stages, which is consistent with its predicted defensive role. The small ncRNA was also upregulated when comparing biotrophy to the in vitro mycelia. This is also accurate if we consider that Meijer and collaborators (2014) discussed that, even though their data cannot shed extra light on the function of these proteins, the data can certainly support the idea that these proteins are expressed during hyphal growth, which can also be encountered during biotrophy to a lesser extent. The berberine-like protein gene was upregulated during biotrophy, which supports the idea that small ncRNAs may only partially regulate the expression of the genes involved in the infection processes.
Finally, small ncRNAs associated with ATP-binding cassette (ABC) transporters (also listed in Table 1) had variable expression. They were initially upregulated, then downregulated and then again upregulated, which concurs with a known pattern characteristic of the transition from biotrophy to necrotrophy. This is because of the response of P. infestans to host defenses. Since common host defenses include the production of reactive oxygen species (ROS) and ABC transporters are involved in detoxification , it is reasonable to expect that their expression increases as the infection progresses towards the necrotrophic stage.
The differential expression found for these small ncRNAs indicates that the ABC transporter genes are expected to present an initial downregulation during biotrophy, then an upregulation during the transition from biotrophy to necrotrophy and then a downregulation again during necrotrophy (Table 1). This behavior allows the pathogen to protect itself from possible plant host defenses. Interestingly, our qRT-PCR experiments confirmed an initial downregulation of ABC transporter #1 during biotrophy when compared to the in vitro mycelia. Subsequently, an increase in its expression to a level similar to that of the in vitro mycelia (at 48 hpi) and then an upregulation during necrotrophy were observed. Although this expression pattern is not exactly as expected, it is again partially supported by the role of the small ncRNAs as regulators of gene expression. As with the haustorium-specific membrane protein gene, ABC transporter #2 did not demonstrate differential expression in the qRT-PCR assays. Again, testing a larger number of ATP-binding transporters and specific haustorium membrane proteins might be necessary to understand their expression profiles with more precision.
Similar studies whose aim is to understand more about the biology of small ncRNAs in Phytophthora species have also included validation steps to evaluate if the expression of the genes is consistent with the expression predicted based on the expression profiles of the small ncRNAs. These studies usually focus on one or a few small ncRNAs or miRNAs and test their expression in a very detailed manner. Luan and collaborators (2016) evaluated a specific miRNA called MIR1918 and then validated its expression via qRT-PCR experiments. Moreover, Cui and collaborators (2014) evaluated the expression of four micro RNAs using a qRT-PCR-based approach. The first of these studies was able to prove that the miRNA seemed to have a negative effect on the expression of the associated gene , and the second was able to corroborate that the miRNA expression levels were as predicted by the bioinformatics analyses .
Jia and collaborators (2017) were interested in evaluating a broader amount of small ncRNAs. To do this, they performed transcriptomic analyses, mainly RNA-Seq, and searched for the presence or absence of the transcripts of genes associated to the small ncRNAs. This allowed them to test for the small ncRNAs’ possible effect in a much more massive way. They also concluded that while 25–26 nt small ncRNAs seem to regulate gene silencing in Phytophthora spp., those small ncRNAs of 21 nt do not seem to participate in this process or at least they do not seem to be the sole regulatory mechanism mediating it . This could also explain why our results indicate a clear association between the small ncRNAs and their correspondent genes in some cases while in others it is not that clear. Also, it is important to consider that small ncRNAs may also be acting indirectly on the expression of genes involved in the process of infection by the regulation of the expression of other genes whose resultant proteins may promote or inhibit the infection’s progression .
Interestingly, several differentially expressed small ncRNAs were associated with genes encoding putative uncharacterized proteins. Future processing of these sequences, possibly by means of a conserved domain analysis search, could be implemented to further investigate their function. As expected, most of the small ncRNAs found to be significantly differentially expressed were identified from comparisons that involved data from the mycelia grown in culture media. This is most likely because, in this case, P. infestans is not exposed to the host, and thus,it is expected that its regulatory processes would be very different from those in any of the other data sets tested.
The presence of some tRNAs, pseudo tRNAs, and one rRNA among the small ncRNAs that showed statistically significant differential expression was not expected due to the removal of these types of RNAs in the implemented pipeline (blue bar in Figure 1). However, the tRNA and rRNA repositories to which the reads were aligned were the ones that performed best among several that were tested. Furthermore, it is important to consider that the genome of P. infestans is highly repetitive  and therefore contains regions that might not be aligning completely, thus hindering the removal of these contaminant sequences.