*2.3. Functional Analysis of Defense Hormone Gene Sequences between A. philoxiroides and A. sessilis*

Six hormones and responsive gene sequences of both invasive and native plants from our study (Table S3) were searched in the NCBI nucleotide database and their identity was checked against other plant species in the Amaranthaceae (Table S4). The output of each gene sequence was obtained to predict a high confidence protein coding sequence from six reading frames (Table S4). Furthermore, a gene from each hormone (*PAL*, *JAR1*, and *EIN3*) was selected on the basis of core signaling component for detailed functional analysis in both *A. philoxiroides* and *A. sessilis*. Each sequence was assembled to a single long contig to predict protein coding genes along with mRNA and the amino acid translation (Table S5). We aimed to compare each gene sequence with other closely related species in Amaranthaceae, and also to compare between *A. philoxiroides* and *A. sessilis* (Figures S2–S5). A comparative phylogenetic analysis of the three hormone genes revealed high conservation within Amaranthaceae (including *Beta vulgaris* and *Spinacia oleracea*, Figure S2). We predicted the conserved domains and motifs in each gene sequence and analyzed how conserved they were across species (see Figure S3 for motifs and Figure S4 for domains). We found that multiple sequence alignments of each amino acid sequence showed high levels of gene conservation between *A. philoxiroides* and *A. sessilis*, and high conservation between the two study species and other related species (Figure S5).

#### *2.4. R. solani Suppresses Jasmonic Acid Signaling for Disease Promotion in A. philoxiroides*

To examine the role of JA-signaling, expression levels of three JA-dependent transcripts, *LOX* (Lipoxygenase), *JAR1* (JA amido synthetase 1), and *PR6* (Proteinase inhibitor), were tested in *A. philoxiroides* following inoculation with *R. solani*. We found the expressions of *JAR1* and *PR6* were reduced in *A. philoxiroides*, whereas *LOX* expression was inconsistent at each time after inoculation (Figure 6a–c). At 6 hpi, all three (*LOX*, *JAR1,* and *PR6*) levels had increased (Figure 6a–c; Table S1). Plants subjected to the MeJA hormone pretreatment also showed reduced expressions across all three JA genes at all time intervals (Figure 6d–f). Overall, our results suggest that JA signaling may be responsible for resistance to *R. solani* in *A. philoxiroides* because JA was induced earlier (up to 6 hpi). During disease progression (after 48 hpi), *R. solani* may overcome JA-resistance signaling by suppressing the defensive responses in *A. philoxiroides* (Figure 6).

**Figure 6.** qPCR analysis of JA-dependent gene expression in *A. philoxiroides*. Four-week-old plants were infected with *R. solani* (**a**–**c**), or sprayed with MeJA pretreatment before being inoculated with *R. solani* (**d**–**f**). Samples were harvested for RNA extractions at the indicated time points (0 to 96 hours). 0 h is the un-inoculated control. Relative expression of JA responsive genes: *JAR1* (**a** and **d**), *PR6* (**b** and **e**), *LOX* (**c**,**f**) were tested with specific primers for *A. philoxiroides* as described in the Methods and Supplementary Table S2. Values represent means ± SE from three biological replicates.

#### *2.5. Salicylic Acid and Ethylene Signaling Enhances Disease Susceptibility in A. philoxiroides*

To determine the role of SA in plant defense response, we tested expression levels of two SA transcripts in *A. philoxiroides*: *PAL* and *PR3* (Supplementary Table S3). We found that the expression of both transcripts consistently increased at each time interval, with the exception of 6 hpi (where expression decreased, Figure 7a,b). The reduced expression at 6 hpi was in contrast to the positive induction of JA transcripts (Figure 6a–c). After inoculation, both *PAL* and *PR3* were also induced in SA pretreated plants (Figure 7d, e). However, *PR3* showed very high levels of expression (up to 100-fold at 48 hpi, Figure 7e and Table S1), suggesting that SA promotes disease development in *A. philoxiroides* following inoculation with *R. solani*.

**Figure 7.** qPCR analysis of SA and ET-responsive gene expression in *A. philoxiroides*. Four-week-old plants were infected with *R. solani* (**a**–**c**), or were sprayed prior to *R. solani* inoculations with SA pretreatment (**d**,**e**) or ET pretreatment (**f**). Samples were harvested for RNA extractions at the indicated time points after the inoculations. 0 h is the un-inoculated control. qPCR was performed with specific primers for SA-*PAL* (**a**,**d**), *PR3* (**b**,**e**), ET-*EIN3* (**c**,**f**) and *Actin* (control) as described in the Methods and shown in Supplementary Table S2. Values represent means ±SE from three biological replicates.

The ET transcript, *EIN3*, displayed a minor increase in expression over time in both the *R. solani* infected and ET-pretreatment samples (Figure 7c,f). Specifically, the expression of *EIN3* was reduced at 6 hpi (similar to SA, Figure 7a). Overall, the results suggest ET-signaling may act synergistically with SA, thereby promoting disease susceptibility in *A. philoxiroides*.

#### *2.6. Signaling Cross-Talk between SA, JA and ET in A. philoxiroides during Interactions with R. solani*

We investigated the cross-talk between hormone signaling pathways in response to *R. solani* infection and hormone pretreatments. We observed that JA (*LOX, JAR1*, and *PR6*) and SA (*PAL* and *PR3*) transcripts had prominent antagonistic cross-talk in *A. philoxiroides* at both earlier (at 6 hpi) and later (>24 hpi) time intervals following *R. solani* inoculations (Figures 6a–c and 7a,b). In addition, MeJA pretreated *A. philoxiroides* showed decreased expressions in all three JA transcripts (Figure 6d–f), and also displayed higher expression levels of SA transcripts (*PAL* and *PR3*) (Figure 8a,b and Table S1). These results indicate a clear antagonistic cross-talk between SA and JA pathways in *A. philoxiroides* after inoculation with *R. solani*.

**Figure 8.** Signaling cross-talk analysis between SA, JA and ET-responsive gene expressions during hormone pretreatments, before *R. solani* inoculations in *A. philoxiroides*. Four-week-old plants were sprayed with MeJA (**a**, **b** and **d**) and SA (**c**). Samples were harvested for RNA extractions at the indicated time points. Un-inoculated leaves were used as a control (0 h). qRT-PCR was performed with specific primers for SA-*PAL* (**a**), *PR3* (**b**), ET-*EIN3* (**c**,**d**) and *Actin* (control) as described in the Methods and shown in Supplementary Table S2. Values represent means ± SE from three biological replicates.

As described above for ET-*EIN3*, gene expression was induced in both treatments in *A. philoxiroides* (Figure 4c, f). In addition, we also tested the expression of *EIN3* with other hormone pretreatments in *A. philoxiroides*. For example, SA pretreated plants induced *EIN3* expression similar to the SA-*PAL* and *PR3* transcripts (Figure 8c). In contrast, MeJA pretreated plants showed a decrease in *EIN3* expression, similar to the JA (*LOX*, *JAR1*, and *PR6*) transcripts (Figure 8d). The differential expression of *EIN3* to SA and MeJA suggests that ET may be regulated by both hormones depending on the type of infection and treatment. Other combinations of hormone signaling and their cross-talk gene expressions are presented in Supplementary Table S1.

To further test whether *R. solani* influences the hormones (SA, JA and ET) during pathogenesis in *A. philoxiroides*, we quantified the endogenous contents of each hormone in *R. solani* infected plants at each time interval (0, 6, 12, 24, 48, 72 and 96 hpi) using ELISA. We detected a 6.9-fold higher SA content (Figure S6a) and a 2.2-fold higher JA content (Supplementary Figure S6b) at 96 hpi compared to the control un-inoculated samples. ET was at a moderate level (about 3.2-fold higher) compared to both SA and JA in the infected plants (Supplementary Figure S6c). The levels of each hormone increased, as time since infection increased (Supplementary Figure S6).

#### *2.7. R. solani Induced Resistance Trade-O*ff*s between SA and JA-Signaling in A. philoxiroides*

To identify and correlate whether hormonal cross-talk provides signals to adjacent leaves from local infected tissues, we examined the resistance trade-offs between SA and JA-signaling in *A. philoxiroides*. Our findings suggest that SA was induced during *R. solani* pathogenesis, which initiated antagonistic cross-talk to JA at the local infected leaves in *A. philoxiroides* (Figures 6 and 7). Investigating neighboring leaves for this cross-talk may provide information regarding how trade-off signals are regulated during infection in invasive *A. philoxiroides*. Therefore, we quantified the expression levels of all six genes in neighboring un-inoculated leaves across all time intervals. We found that JA-*JAR1* showed reduced expressions at each time interval (Figure 9a). In contrast, increased expression of SA-*PAL* from 48 hpi was detected (Figure 9b). The cross-talk between these transcripts in the neighboring un-inoculated

leaf was similar to the infected leaves. However, other transcripts of JA-*LOX* and *PR6* were induced in the same plants (Figure 9c,d), while SA-*PR3* was much reduced (Figure 9e). These findings suggest that the key signaling component of SA (*PAL*) and JA (*JAR1*) engage in antagonistic cross-talk during pathogenesis. The ET-*EIN3* expression was reduced at each time interval (Figure 9f).

**Figure 9.** Expression analysis of SA, JA and ET-responsive genes for systemic acquired resistance tests in *A*. *philoxiroides*. Four-week-old *A. philoxiroides* were infected with *R. solani* and samples of healthy un-inoculated leaves were collected for RNA extractions (at time intervals from 0 to 96 hours). 0 h is from the samples of plant completely un-infected for control. Relative expression of *JAR1* (**a**), *PAL* (**b**), *LOX* (**c**), *PR6* (**d**), *PR3* (**e**) and *EIN3* (**f**) with *Actin* (control) genes were tested using specific primers at the indicated time intervals. Values represent means ± SE from three biological replicates.
