Transcriptome analysis has recently been addressed as an alternative to provide insights into plant pathogenicity mechanisms used by R. necatrix [12, 13]. However, neither of the two studies was carried out using R. necatrix directly interacting with a host. This current study fills this gap, obtaining and analyzing the transcriptomes of this fungus during infection of avocado roots and comparing it with that obtained from the fungus cultured in rich medium.
The number of predicted genes (12,104) is congruent with data from previous transcriptomes obtained from R. necatrix (10,616; ), as well as other plant pathogenic Ascomycota, such as Fusarium graminearum (13,332 genes; ), Valsa mali (13,046 genes; ), or Magnaporte oryzae (11,101 genes; ), suggesting an overall good assembly quality.
When comparing gene expression profiles between R. necatrix infecting avocado roots or growing on PDA medium, a number of transcripts were related with major fungal traits involved in the interaction with the host, among others, CWDE , production of toxic compounds and detoxification of those produced by the host, or potential effectors.
Phytopathogenic fungi usually produce numerous extracellular enzymes in order to penetrate the host tissue, being cell wall hydrolases and pectinases the most important ones . The high number of CWDE over-expressed during the infection process correlates with previous visualization studies of R. necatrix hyphae that directly penetrate through the avocado root cells . In addition, five putative proteases were also identified. Interestingly, gene expression studies carried out on avocado revealed that three protease inhibitors were highly over-expressed in tolerant rootstocks to R. necatrix following inoculation with the pathogen but not in susceptible genotypes10]. This finding suggests that these proteases, up-regulated in R. necatrix during the infection process, could play an important role in degrading basal defense proteins on susceptible avocado roots, however, future experiments need to be carried out to confirm this hypothesis.
Several studies support the idea that R. necatrix produce toxins that are likely responsible for the symptoms observed in the aerial parts of the plant [26, 27]. Cytochalasin E and rosnecatrone toxins produced by R. necatrix [28, 29] are believed to be involved in the onset of disease symptoms in young apple shoots and detached apple leaves . Shimizu et al., , identified the cytochalasin biosynthetic gene cluster, containing fourteen genes, within a 36 kb region of the R. necatrix strain W97 genome. In the present study, only one gene (putative aflatoxin B1 aldehyde reductase protein) conforming the putative cytochalasin cluster was highly up-regulated, while it was down-regulated in transcriptomic analyses carried out in the hypovirulent R. necatrix strain  (Additional file 2: Table S2). Taking this into consideration, this gene could play an important role in the pathogenicity of R. necatrix CH53on avocado roots, however the role of the cytochalasin E in virulence remains unclear as suggested by other authors . Four more genes related with the production of fungal toxins were up-regulated during the infection process, two of them (putative sterigmatocystin 8-O-methyltransferase and the averantin oxidoreductase) had been previously described to be involved in aflatoxin biosynthesis . Aflatoxins are considered as the most toxic and carcinogenic compounds among the known mycotoxins and 25 clustered genes have been reported to be involved in its biosynthesis [31, 32]. Although the expression of other genes potentially involved in aflatoxin biosynthesis was not observed and no aflatoxin production, even at minimum concentration (< 1 g/Kg), was detected in wheat grains infected with R necatrix (data not shown), future studies should address the detection of this compound on infected roots due to its high toxigenic nature.
As other necrotrophic pathogens, R. necatrix seems to have adapted mechanisms to detoxify host metabolites that can interfere with its virulence . Nineteen genes potentially involved in detoxification of antimicrobial compounds were significantly over-expressed. Interestingly, SAMD00023353_12800020 and SAMD00023353_3200110, both repressed in the hypovirulent R. necatrix strain , showed homology to genes previously described to be involved in detoxification of phytoalexins. The importance of phytoalexin degradation ability in pathogenesis has been proved through transformation experiments . To date, no phytoalexin production has been reported in ‘Dusa’ avocado rootstocks however, mutation experiments of these two genes would be of great interest to reveal their role in degradation of possible fungal toxic compounds produced by avocado roots.
Other contigs were related to transport mechanisms by which endogenous and exogenous toxicants can be secreted. Two major classes of transporter proteins were represented in R. necatrix DEGs such as ABC and MFS transporters. Members of both classes can have broad and overlapping substrate specificities for toxic compounds and have been considered as a “first-line fungus defense barrier” .
Some necrotrophs are also able to influence host phytohormone levels or employ their own hormone biosynthesis machinery thereby disrupting defense signaling [24, 36–41]. Two genes involved in gibberellin biosynthesis, GA4 desaturase family protein and Gibberellin 20-oxidase, were up-regulated during the infection process. Role of GAs in plant-pathogen interactions is not well known ; i.e., Studt et al.  showed the positive relation between GA production and bakanae disease in rice while Manka  found no correlation between GA production and pathogenesis of Fusarium.
Throughout the infection process, fungi can actively manipulate host cellular machinery in order to suppress defenses and/or aid disease progression throughout the release of the so-called ‘effector´ proteins . These effectors are usually secreted proteins that act at the host cell surface  or are taken up by the plant cell and act internally . In this investigation, a total of 23 genes were predicted to be effectors (with probability above 60%), among which 19 encoded for hypothetical proteins and 10 were predicted as apoplastic effectors, being their place of action the interphase between the hyphae and the host cell. One of the predicted effectors, showed homology to the Lysm1 effector of Penycilium expansum. Lysm-containing proteins have been proposed to be involved in binding and sequestering chitin oligosaccharides in order to prevent elicitation of host immune responses  and/or to protect fungal hyphae against chitinases secreted by competitors . In this sense, the expression of this effector during R. necatrix infection correlates with previous studies in which the overexpression of chitinases on susceptible avocado rootstocks/R. necatrix interaction, was reported . Finally, other contig showed homology with the previously described Blumeria graminis effector gene BEC1040, which reduces haustoria formation in barley powdery mildew when silenced . These results confirm previous observations by , in which BEC1040 homologous effectors in the virulent R. necatrix strain KACC40445 were found.